AUTHOR TITLE JOURNAL LANGUANGE DOCTYPE KEYWORDS ABSTRACT

AFFILIATION EMAIL DOI PUBYEAR PUBVOL PUBISSUE FPAGE LPAGE

ARTNUMBER PAGENUM
Wagner, Gabriel; Totaro, Massimo; Volland, André; Lipp, Michaela; Saiger, Sabine;
Lichtenegger, Sabine; Forstner, Patrick; von Laer, Dorothee; Oberdorfer, Gustav;
Steinmetz, Ivo A Novel High-Throughput Nanopore-Sequencing-Based Strategy for
Rapid and Automated S-Protein Typing of SARS-CoV-2 VariantsViruses EN
Communication SARS-CoV-2; next-generation sequencing; vaccine escape; S-
protein; nanopore; surveillance; typing Rapid molecular surveillance of SARS-CoV-
2 S-protein variants leading to immune escape and/or increased infectivity is of
utmost importance. Among global bottlenecks for variant monitoring in diagnostic
settings are sequencing and bioinformatics capacities. In this study, we aimed to
establish a rapid and user-friendly protocol for high-throughput S-gene sequencing
and subsequent automated identification of variants. We designed two new primer
pairs to amplify only the immunodominant part of the S-gene for nanopore
sequencing. Furthermore, we developed an automated “S-Protein-Typer”
tool that analyzes and reports S-protein mutations on the amino acid level
including a variant of concern indicator. Validation of our primer panel using
SARS-CoV-2-positive respiratory specimens covering a broad Ct range showed
successful amplification for 29/30 samples. Restriction to the region of interest
freed sequencing capacity by a factor of 12–13, compared with whole-genome
sequencing. Using either the MinION or Flongle flow cell, our sequencing strategy
reduced the time required to identify SARS-CoV-2 variants accordingly. The S-
Protein-Typer tool identified all mutations correctly when challenged with our
sequenced samples and 50 deposited sequences covering all VOCs (December 2021). Our
proposed S-protein variant screening offers a simple, more rapid, and low-cost
entry into NGS-based SARS-CoV-2 analysis, compared with current whole-genome
approaches. Diagnostic and Research Institute of Hygiene, Microbiology and
Environmental Medicine, Medical University of Graz, 8010 Graz, Austria
gabriel.wagner-lichtenegger@medunigraz.at; massimo.totaro@tugraz.at;
andre.volland@i-med.ac.at; michaela.lipp@medunigraz.at;
sabine.saiger@medunigraz.at; sabine.lichtenegger@medunigraz.at;
patrick.forstner@medunigraz.at; dorothee.von-laer@i-med.ac.at;
gustav.oberdorfer@tugraz.at; ivo.steinmetz@medunigraz.at 10.3390/v13122548 2021
13 12 - - 2548 -
Macqueen, Daniel; Eve, Oliver; Gundappa, Manu; Daniels, Rose; Gallagher, Michael;
Alexandersen, Svein; Karlsen, Marius Genomic Epidemiology of Salmonid
Alphavirus in Norwegian Aquaculture Reveals Recent Subtype-2 Transmission Dynamics
and Novel Subtype-3 Lineages Viruses EN Article genomic epidemiology;
genomic surveillance; viral genomics; aquaculture; salmonid alphavirus; pancreas
disease Viral disease poses a major barrier to sustainable aquaculture, with
outbreaks causing large economic losses and growing concerns for fish welfare.
Genomic epidemiology can support disease control by providing rapid inferences on
viral evolution and disease transmission. In this study, genomic epidemiology was
used to investigate salmonid alphavirus (SAV), the causative agent of pancreas
disease (PD) in Atlantic salmon. Our aim was to reconstruct SAV subtype-2 (SAV2)
diversity and transmission dynamics in recent Norwegian aquaculture, including the
origin of SAV2 in regions where this subtype is not tolerated under current
legislation. Using nanopore sequencing, we captured ~90% of the SAV2 genome for n =
68 field isolates from 10 aquaculture production regions sampled between 2018 and
2020. Using time-calibrated phylogenetics, we infer that, following its
introduction to Norway around 2010, SAV2 split into two clades (SAV2a and 2b)
around 2013. While co-present at the same sites near the boundary of Møre og
Romsdal and Trøndelag, SAV2a and 2b were generally detected in non-
overlapping locations at more Southern and Northern latitudes, respectively. We
provide evidence for recent SAV2 transmission over large distances, revealing a
strong connection between Møre og Romsdal and SAV2 detected in 2019/20 in
Rogaland. We also demonstrate separate introductions of SAV2a and 2b outside the
SAV2 zone in Sognefjorden (Vestland), connected to samples from Møre og
Romsdal and Trøndelag, respectively, and a likely 100 km Northward
transmission of SAV2b within Trøndelag. Finally, we recovered genomes of
SAV2a and SAV3 co-infecting single fish in Rogaland, involving novel SAV3 lineages
that diverged from previously characterized strains >25 years ago. Overall, this
study demonstrates useful applications of genomic epidemiology for tracking viral
disease spread in aquaculture. The Roslin Institute and Royal (Dick) School of
Veterinary Studies, The University of Edinburgh, Edinburgh EH25 9RG, UK
daniel.macqueen@roslin.ed.ac.uk; oliver.eve@ed.ac.uk;
manu.gundappa@roslin.ed.ac.uk; rose.daniels@roslin.ed.ac.uk;
mgallagher@bionanogenomics.com; svein.alexandersen@zoetis.com;
marius.karlsen@zoetis.com 10.3390/v13122549 2021 13 12 - - 2549
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Li, Bing; Zhang, Xueli; Liu, Zhiquan; Wang, Lulin; Song, Liping; Liang, Xiaomei;
Dou, Shengwei; Tu, Jinxing; Shen, Jinxiong; Yi, Bin; Wen, Jing; Fu, Tingdong; Dai,
Cheng; Gao, Changbin; Wang, Aihua; Ma, Chaozhi Genetic and Molecular
Characterization of a Self-Compatible Brassica rapa Line Possessing a New Class II
S Haplotype Plants EN Article Brassica rapa; self-incompatibility/self-
compatibility; S haplotype; S locus receptor kinase (SRK); S locus cysteine-rich
protein (SCR); amino acid variations Most flowering plants have evolved a
self-incompatibility (SI) system to maintain genetic diversity by preventing self-
pollination. The Brassica species possesses sporophytic self-incompatibility (SSI),
which is controlled by the pollen- and stigma-determinant factors SP11/SCR and SRK.
However, the mysterious molecular mechanism of SI remains largely unknown. Here, a
new class II S haplotype, named BrS-325, was identified in a pak choi line
‘325’, which was responsible for the completely self-compatible
phenotype. To obtain the entire S locus sequences, a complete pak choi genome was
gained through Nanopore sequencing and de novo assembly, which provided a good
reference genome for breeding and molecular research in B. rapa. S locus
comparative analysis showed that the closest relatives to BrS-325 was BrS-60, and
high sequence polymorphism existed in the S locus. Meanwhile, two duplicated SRKs
(BrSRK-325a and BrSRK-325b) were distributed in the BrS-325 locus with opposite
transcription directions. BrSRK-325b and BrSCR-325 were expressed normally at the
transcriptional level. The multiple sequence alignment of SCRs and SRKs in class II
S haplotypes showed that a number of amino acid variations were present in the
contact regions (CR II and CR III) of BrSCR-325 and the hypervariable regions (HV I
and HV II) of BrSRK-325s, which may influence the binding and interaction between
the ligand and the receptor. Thus, these results suggested that amino acid
variations in contact sites may lead to the SI destruction of a new class II S
haplotype BrS-325 in B. rapa. The complete SC phenotype of ‘325’ showed
the potential for practical breeding application value in B. rapa. National
Sub-Center of Rapeseed Improvement in Wuhan, National Key Laboratory of Crop
Genetic Improvement, College of Plant Science and Technology, Huazhong Agricultural
University, Wuhan 430070, China 18735424247@163.com; zywweirui@sina.cn;
lzq0826@163.com; 15110670184@163.com; lp19871120@126.com; 15071303711@163.com;
doushengwei@webmail.hzau.edu.cn; tujx@mail.hzau.edu.cn; jxshen@mail.hzau.edu.cn;
yibin@mail.hzau.edu.cn; wenjing@mail.hzau.edu.cn; futing@mail.hzau.edu.cn;
cdai@mail.hzau.edu.cn; gaocb1983@163.com; wangaihualt@163.com;
yuanbeauty@mail.hzau.edu.cn 10.3390/plants10122815 2021 10 12 - -
2815 -
Kirov, Ilya; Polkhovskaya, Ekaterina; Dudnikov, Maxim; Merkulov, Pavel; Vlasova,
Anastasia; Karlov, Gennady; Soloviev, Alexander Searching for a Needle in a
Haystack: Cas9-Targeted Nanopore Sequencing and DNA Methylation Profiling of Full-
Length Glutenin Genes in a Big Cereal Genome Plants EN Communication
nanopore sequencing; Cas9-enrichment; triticale; glutenin genes; DNA
methylation Sequencing and epigenetic profiling of target genes in plants are
important tasks with various applications ranging from marker design for plant
breeding to the study of gene expression regulation. This is particularly
interesting for plants with big genome size for which whole-genome sequencing can
be time-consuming and costly. In this study, we asked whether recently proposed
Cas9-targeted nanopore sequencing (nCATS) is efficient for target gene sequencing
for plant species with big genome size. We applied nCATS to sequence the full-
length glutenin genes (Glu-1Ax, Glu-1Bx and Glu-1By) and their promoters in
hexaploid triticale (X Triticosecale, AABBRR, genome size is 24 Gb). We showed that
while the target gene enrichment per se was quite high for the three glutenin genes
(up to 645×), the sequencing depth that was achieved from two MinION
flowcells was relatively low (5–17×). However, this sequencing depth
was sufficient for various tasks including detection of InDels and single-
nucleotide variations (SNPs), read phasing and methylation profiling. Using nCATS,
we uncovered SNP and InDel variation of full-length glutenin genes providing useful
information for marker design and deciphering of variation of individual Glu-1By
alleles. Moreover, we demonstrated that glutenin genes possess a ‘gene-
body’ methylation epigenetic profile with hypermethylated CDS part and
hypomethylated promoter region. The obtained information raised an interesting
question on the role of gene-body methylation in glutenin gene expression
regulation. Taken together, our work disclosures the potential of the nCATS
approach for sequencing of target genes in plants with big genome size.
Laboratory of Marker-Assisted and Genomic Selection of Plants, All-Russia
Research Institute of Agricultural Biotechnology, Timiryazevskaya Str. 42, 127550
Moscow, Russia kirovez@gmail.com; eynzeynkreyn@gmail.com;
max.dudnikov.07@gmail.com; paulmerkulov97@gmail.com; vlasova.nactia@yandex.ru;
karlovg@gmail.com; a.soloviev70@gmail.com 10.3390/plants11010005 2021 11 1
- - 5 -
Eltokhy, Mohamed; Saad, Bishoy; Eltayeb, Wafaa; Yahia, Ibrahim; Aboshanab, Khaled;
Ashour, Mohamed Exploring the Nature of the Antimicrobial Metabolites Produced by
Paenibacillus ehimensis Soil Isolate MZ921932 Using a Metagenomic Nanopore
Sequencing Coupled with LC-Mass Analysis Antibiotics EN Article
Paenibacillus ehimensis; LC/MS; metagenomics; β-lactone; petrobactin;
tridecaptin; locillomycin; polymyxin; terpene; polyketide The continuous emergence
of multidrug-resistant (MDR) pathogens poses a global threat to public health.
Accordingly, global efforts are continuously conducted to find new approaches to
infection control by rapidly discovering antibiotics, particularly those that
retain activities against MDR pathogens. In this study, metagenomic nanopore
sequence analysis coupled with spectroscopic methods has been conducted for rapid
exploring of the various active metabolites produced by Paenibacillus ehimensis
soil isolate. Preliminary soil screening resulted in selection of a Gram-positive
isolate identified via 16S ribosomal RNA gene sequencing as Paenibacillus ehimensis
MZ921932. The isolate showed a broad range of activity against MDR Gram-positive,
Gram-negative, and Candida spp. A metagenomics sequence analysis of the soil sample
harboring Paenibacillus ehimensis isolate MZ921932 (NCBI GenBank accession
PRJNA785410) revealed the presence of conserved biosynthetic gene clusters of
petrobactin, tridecaptin, locillomycin (β-lactone), polymyxin, and macrobrevin
(polyketides). The liquid chromatography/mass (LC/MS) analysis of the Paenibacillus
ehimensis metabolites confirmed the presence of petrobactin, locillomycin, and
macrobrevin. In conclusion, Paenibacillus ehimensis isolate MZ921932 is a promising
rich source for broad spectrum antimicrobial metabolites. The metagenomic nanopore
sequence analysis was a rapid, easy, and efficient method for the preliminary
detection of the nature of the expected active metabolites. LC/MS spectral analysis
was employed for further confirmation of the nature of the respective active
metabolites. Department of Microbiology, Faculty of Pharmacy, Misr
International University (MIU), Cairo 19648, Egypt
mohammad.ashraf@miuegypt.edu.eg; bishoyth@hitssolutions.com;
wafaa.eltayeb@miuegypt.edu.eg; ihussein@kku.edu.sa;
aboshanab2012@pharma.asu.edu.eg; seifashour@hotmail.com
10.3390/antibiotics11010012 2021 11 1 - - 12 -
Athanasopoulou, Konstantina; Boti, Michaela; Adamopoulos, Panagiotis; Skourou,
Paraskevi; Scorilas, Andreas Third-Generation Sequencing: The Spearhead towards
the Radical Transformation of Modern Genomics Life EN Review long-read
sequencing; PacBio sequencing; nanopore sequencing; single-molecule real-time
sequencing; targeted DNA sequencing; direct RNA sequencing; metagenomics;
epigenomics; epitranscriptomics Although next-generation sequencing (NGS)
technology revolutionized sequencing, offering a tremendous sequencing capacity
with groundbreaking depth and accuracy, it continues to demonstrate serious
limitations. In the early 2010s, the introduction of a novel set of sequencing
methodologies, presented by two platforms, Pacific Biosciences (PacBio) and Oxford
Nanopore Sequencing (ONT), gave birth to third-generation sequencing (TGS). The
innovative long-read technologies turn genome sequencing into an ease-of-handle
procedure by greatly reducing the average time of library construction workflows
and simplifying the process of de novo genome assembly due to the generation of
long reads. Long sequencing reads produced by both TGS methodologies have already
facilitated the decipherment of transcriptional profiling since they enable the
identification of full-length transcripts without the need for assembly or the use
of sophisticated bioinformatics tools. Long-read technologies have also provided
new insights into the field of epitranscriptomics, by allowing the direct detection
of RNA modifications on native RNA molecules. This review highlights the
advantageous features of the newly introduced TGS technologies, discusses their
limitations and provides an in-depth comparison regarding their scientific
background and available protocols as well as their potential utility in research
and clinical applications. Department of Biochemistry and Molecular Biology,
Faculty of Biology, National and Kapodistrian University of Athens, 15701 Athens,
Greece konnath@biol.uoa.gr; miboti@biol.uoa.gr; padamopoulos@biol.uoa.gr;
pskourou@biol.uoa.gr; ascorilas@biol.uoa.gr 10.3390/life12010030 2021 12
1 - - 30 -
Vacca, Davide; Fiannaca, Antonino; Tramuto, Fabio; Cancila, Valeria; La Paglia,
Laura; Mazzucco, Walter; Gulino, Alessandro; La Rosa, Massimo; Maida, Carmelo;
Morello, Gaia; Belmonte, Beatrice; Casuccio, Alessandra; Maugeri, Rosario;
Iacopino, Gerardo; Balistreri, Carmela; Vitale, Francesco; Tripodo, Claudio; Urso,
Alfonso Direct RNA Nanopore Sequencing of SARS-CoV-2 Extracted from Critical
Material from Swabs Life EN Article MinION; direct RNA nanopore
sequencing; SARS-CoV-2; COVID-19; swab In consideration of the increasing
prevalence of COVID-19 cases in several countries and the resulting demand for
unbiased sequencing approaches, we performed a direct RNA sequencing (direct RNA
seq.) experiment using critical oropharyngeal swab samples collected from Italian
patients infected with SARS-CoV-2 from the Palermo region in Sicily. Here, we
identified the sequences SARS-CoV-2 directly in RNA extracted from critical samples
using the Oxford Nanopore MinION technology without prior cDNA retrotranscription.
Using an appropriate bioinformatics pipeline, we could identify mutations in the
nucleocapsid (N) gene, which have been reported previously in studies conducted in
other countries. In conclusion, to the best of our knowledge, the technique used in
this study has not been used for SARS-CoV-2 detection previously owing to the
difficulties in the extraction of RNA of sufficient quantity and quality from
routine oropharyngeal swabs. Despite these limitations, this approach provides the
advantages of true native RNA sequencing and does not include amplification steps
that could introduce systematic errors. This study can provide novel information
relevant to the current strategies adopted in SARS-CoV-2 next-generation
sequencing. Department of Surgical, Oncological and Oral Sciences, University of
Palermo, 90127 Palermo, Italy davide.vacca@unipa.it; antonino.fiannaca@icar.cnr.it;
fabio.tramuto@unipa.it; valeria.cancila@unipa.it; laura.lapaglia@icar.cnr.it;
walter.mazzucco@unipa.it; alessandro.gulino@cogentech.it;
massimo.larosa@icar.cnr.it; carmelo.maida@unipa.it; gaia.morello@unipa.it;
beatrice.belmonte@unipa.it; alessandra.casuccio@unipa.it;
rosario.maugeri1977@gmail.com; gerardo.iacopino@gmail.com;
carmelarita.balistreri@unipa.it; francesco.vitale@unipa.it;
claudio.tripodo@unipa.it; alfonso.urso@icar.cnr.it 10.3390/life12010069 2022
12 1 - - 69 -
Lüth, Theresa; Laβ, Joshua; Schaake, Susen; Wohlers, Inken; Pozojevic, Jelena;
Jamora, Roland; Rosales, Raymond; Brüggemann, Norbert; Saranza, Gerard; Diesta,
Cid; Schlüter, Kathleen; Tse, Ronnie; Reyes, Charles; Brand, Max; Busch, Hauke;
Klein, Christine; Westenberger, Ana; Trinh, Joanne Elucidating Hexanucleotide
Repeat Number and Methylation within the X-Linked Dystonia-Parkinsonism (XDP)-
Related SVA Retrotransposon in TAF1 with Nanopore Sequencing Genes EN
Article X-linked dystonia-parkinsonism; nanopore sequencing; repeat
motif; CpG methylation Background: X-linked dystonia-parkinsonism (XDP) is an
adult-onset neurodegenerative disorder characterized by progressive dystonia and
parkinsonism. It is caused by a SINE-VNTR-Alu (SVA) retrotransposon insertion in
the TAF1 gene with a polymorphic (CCCTCT)n domain that acts as a genetic modifier
of disease onset and expressivity. Methods: Herein, we used Nanopore sequencing to
investigate SVA genetic variability and methylation. We used blood-derived DNA from
96 XDP patients for amplicon-based deep Nanopore sequencing and validated it with
fragment analysis which was performed using fluorescence-based PCR. To detect
methylation from blood- and brain-derived DNA, we used a Cas9-targeted approach.
Results: High concordance was observed for hexanucleotide repeat numbers detected
with Nanopore sequencing and fragment analysis. Within the SVA locus, there was no
difference in genetic variability other than variations of the repeat motif between
patients. We detected high CpG methylation frequency (MF) of the SVA and flanking
regions (mean MF = 0.94, SD = ±0.12). Our preliminary results suggest only
subtle differences between the XDP patient and the control in predicted enhancer
sites directly flanking the SVA locus. Conclusions: Nanopore sequencing can
reliably detect SVA hexanucleotide repeat numbers, methylation and, lastly,
variation in the repeat motif. Institute of Neurogenetics, University of
Luebeck, 23538 Luebeck, Germany theresa.lueth@neuro.uni-luebeck.de;
joshua.lass@student.uni-luebeck.de; susen.schaake@neuro.uni-luebeck.de;
inken.wohlers@uni-luebeck.de; jelena.pozojevic@neuro.uni-luebeck.de;
rgjamora@up.edu.ph; rlrosales@ust.edu.ph; norbert.brueggemann@neuro.uni-luebeck.de;
gerardsaranza@gmail.com; ciddiesta@gmail.com; kathleen.schlueter@student.uni-
luebeck.de; ronnie.tse@neuro.uni-luebeck.de; charles.reyes@neuro.uni-luebeck.de;
max.brand@student.uni-luebeck.de; hauke.busch@uni-luebeck.de;
christine.klein@neuro.uni-luebeck.de; ana.westenberger@neuro.uni-luebeck.de;
joanne.trinh@neuro.uni-luebeck.de 10.3390/genes13010126 2022 13 1 -
- 126 -
Boysen, Gunnar; Nookaew, Intawat Current and Future Methodology for Quantitation
and Site-Specific Mapping the Location of DNA Adducts Toxics EN
Communication DNA adducts; nanopore; Oxford Nanopore Technology; mass
spectrometry; adductomics; exposome Formation of DNA adducts is a key event for a
genotoxic mode of action, and their presence is often used as a surrogate for
mutation and increased cancer risk. Interest in DNA adducts are twofold: first, to
demonstrate exposure, and second, to link DNA adduct location to subsequent
mutations or altered gene regulation. Methods have been established to quantitate
DNA adducts with high chemical specificity and to visualize the location of DNA
adducts, and elegant bio-analytical methods have been devised utilizing enzymes,
various chemistries, and molecular biology methods. Traditionally, these highly
specific methods cannot be combined, and the results are incomparable. Initially
developed for single-molecule DNA sequencing, nanopore-type technologies are
expected to enable simultaneous quantitation and location of DNA adducts across the
genome. Herein, we briefly summarize the current methodologies for state-of-the-art
quantitation of DNA adduct levels and mapping of DNA adducts and describe novel
single-molecule DNA sequencing technologies to achieve both measures. Emerging
technologies are expected to soon provide a comprehensive picture of the exposome
and identify gene regions susceptible to DNA adduct formation. Department
Environmental and Occupational Health, Fay W. Boozman College of Public Health,
University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
gboysen@uams.edu; inookaew@uams.edu 10.3390/toxics10020045 2022 10 2
- - 45 -
Li, Shuwen; Zhao, Shuxue; Hu, Chunhui; Mao, Chengzhi; Guo, Lizhong; Yu, Hailong;
Yu, Hao Whole Genome Sequence of an Edible Mushroom Stropharia rugosoannulata
(Daqiugaigu) Journal of Fungi EN Article genome; Stropharia
rugosoannulata; mushroom; lignocellulose degradation; CAZymes Stropharia
rugosoannulata, also known as Daqiugaigu in China, is a well-known edible mushroom
that has been widely cultivated in China in recent years. Many studies have focused
on its nutrients, bioactive compounds, and lignin degradation capacity, although
there are few molecular and genetic breeding studies due to the lack of genomic
information. Here, we present the 47.9 Mb genome sequence of an S. rugosoannulata
monokaryotic strain (A15), which has 20 contigs and an N50 of 3.64 Mb, which was
obtained by a combination of Illumina and Nanopore sequencing platforms. Further
analysis predicted 12,752 protein-coding genes, including 486 CAZyme-encoding
genes. Phylogenetic analysis revealed a close evolutionary relationship between S.
rugosoannulata and Hypholoma sublateritium, Psilocybe cyanescens, and Galerina
marginata based on single-copy orthologous genes. Proteomic analysis revealed
different protein expression profiles between the cap and the stipe of the S.
rugosoannulata fruiting body. The proteins of the stipe associated with carbon
metabolism, energy production, and stress-response-related biological processes had
higher abundance, whereas proteins involved in fatty acid synthesis and mRNA
splicing showed higher expression in the cap than in the stipe. The genome of S.
rugosoannulata will provide valuable genetic resources not only for comparative
genomic analyses and evolutionary studies among Basidiomycetes but also for
alleviating the bottlenecks that restrict the molecular breeding of this edible
mushroom. Shandong Provincial Key Laboratory of Applied Mycology, School of Life
Sciences, Qingdao Agricultural University, 700 Changcheng Road, Qingdao 266109,
China swli@qau.edu.cn; 11201011028@stu.ouc.edu.cn; 201201012@qau.edu.cn;
20212106012@stu.qau.edu.cn; 198701007@qau.edu.cn; yuhailong@saas.sh.cn;
yuhao@qau.edu.cn 10.3390/jof8020099 2022 8 2 - - 99 -
Murase, Kazunori Cytolysin A (ClyA): A Bacterial Virulence Factor with Potential
Applications in Nanopore Technology, Vaccine Development, and Tumor Therapy Toxins
EN Review cytolysin A; pore-forming toxin; outer membrane vesicles;
biotechnology application Cytolysin A (ClyA) is a pore-forming toxin that is
produced by some bacteria from the Enterobacteriaceae family. This review provides
an overview of the current state of knowledge regarding ClyA, including the
prevalence of the encoding gene and its transcriptional regulation, the secretion
pathway used by the protein, and the mechanism of protein assembly, and highlights
potential applications of ClyA in biotechnology. ClyA expression is regulated at
the transcriptional level, primarily in response to environmental stressors, and
ClyA can exist stably both as a soluble monomer and as an oligomeric membrane
complex. At high concentrations, ClyA induces cytolysis, whereas at low
concentrations ClyA can affect intracellular signaling. ClyA is secreted in outer
membrane vesicles (OMVs), which has important implications for biotechnology
applications. For example, the native pore-forming ability of ClyA suggests that it
could be used as a component of nanopore-based technologies, such as sequencing
platforms. ClyA has also been exploited in vaccine development owing to its ability
to present antigens on the OMV surface and provoke a robust immune response. In
addition, ClyA alone or OMVs carrying ClyA fusion proteins have been investigated
for their potential use as anti-tumor agents. Department of Microbiology,
Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
murase.kazunori.3x@kyoto-u.ac.jp 10.3390/toxins14020078 2022 14 2
- - 78 -
Amoia, Serafina; Minafra, Angelantonio; Nicoloso, Vittorio; Loconsole, Giuliana;
Chiumenti, Michela A New Jasmine Virus C Isolate Identified by Nanopore
Sequencing Is Associated to Yellow Mosaic Symptoms of Jasminum officinale in Italy
Plants EN Article jasmine; virus; yellow mosaic; high-throughput
sequencing; virus detection; Carlavirus Some plants of Jasminum officinale were
selected in a nursery for investigation of sanitary status of candidate mother
plants before vegetative propagation. The presence of yellow spots and leaf
discoloration symptoms pushed for a generic diagnosis through deep sequencing to
discover systemic pathogens. Either dsRNA or total RNA were extracted and used in
nanopore and Illumina platform for cDNA-PCR, direct RNA and total RNA rRNA-depleted
sequencing. A few single reads obtained by nanopore technology or assembled contigs
gave unequivocal annotation for the only presence of a jasmine virus C (JaVC, a
putative member of genus Carlavirus) isolate. The full-length genome of this
isolate was reconstructed, spanning 8490 nucleotides (nt). This isolate shared
90.9% similarity with coat protein sequences and 84% with the entire ORF1
polyprotein, with the other two available JaVC full genomes, isolated from
infections in J. sambac in Taiwan and China. The overall nucleotide identity shared
by the newly discovered Italian isolate with the Chinese JaVC full genomes was
76.14% (Taiwan) and 75.60% (Fujian). The application of quick nanopore sequencing
for virus discovery was assessed. The identification of the virus in a new
ornamental host species, largely used in gardening, creates a concern for the
potential virus spread and need of testing for production of clean vegetative
material. Institute for Sustainable Plant Protection—National Research Council,
70126 Bari, Italy serena.amoia@ipsp.cnr.it; angelantonio.minafra@ipsp.cnr.it;
vittorio.nicoloso@ipsp.cnr.it; giuliana.loconsole@ipsp.cnr.it;
michela.chiumenti@ipsp.cnr.it 10.3390/plants11030309 2022 11 3 - -
309 -
Shiao, Yih-Horng Promising Assays for Examining a Putative Role of Ribosomal
Heterogeneity in COVID-19 Susceptibility and Severity Life EN Review
translation machinery; SARS-CoV-2; COVID-19; ribosomes; ribosomal
heterogeneity; ribosomal RNAs; ribosomal proteins; assays for RNA sequence and
structure; assays for proteins The heterogeneity of ribosomes, characterized
by structural variations, arises from differences in types, numbers, and/or post-
translational modifications of participating ribosomal proteins (RPs), ribosomal
RNAs (rRNAs) sequence variants plus post-transcriptional modifications, and
additional molecules essential for forming a translational machinery. The ribosomal
heterogeneity within an individual organism or a single cell leads to preferential
translations of selected messenger RNA (mRNA) transcripts over others, especially
in response to environmental cues. The role of ribosomal heterogeneity in SARS-CoV-
2 coronavirus infection, propagation, related symptoms, or vaccine responses is not
known, and a technique to examine these has not yet been developed. Tools to detect
ribosomal heterogeneity or to profile translating mRNAs independently cannot
identify unique or specialized ribosome(s) along with corresponding mRNA
substrate(s). Concurrent characterizations of RPs and/or rRNAs with mRNA substrate
from a single ribosome would be critical to decipher the putative role of ribosomal
heterogeneity in the COVID-19 disease, caused by the SARS-CoV-2, which hijacks the
host ribosome to preferentially translate its RNA genome. Such a protocol should be
able to provide a high-throughput screening of clinical samples in a large
population that would reach a statistical power for determining the impact of a
specialized ribosome to specific characteristics of the disease. These
characteristics may include host susceptibility, viral infectivity and
transmissibility, severity of symptoms, antiviral treatment responses, and vaccine
immunogenicity including its side effect and efficacy. In this study, several
state-of-the-art techniques, in particular, chemical probing of ribosomal
components or rRNA structures, proximity ligation to generate rRNA-mRNA chimeras
for sequencing, nanopore gating of individual ribosomes, nanopore RNA sequencing
and/or structural analyses, single-ribosome mass spectrometry, and microfluidic
droplets for separating ribosomes or indexing rRNAs/mRNAs, are discussed. The key
elements for further improvement and proper integration of the above techniques to
potentially arrive at a high-throughput protocol for examining individual ribosomes
and their mRNA substrates in a clinical setting are also presented. US Patent
Trademark Office, Department of Commerce, Alexandria, VA 22314, USA
yihhorng@aol.com 10.3390/life12020203 2022 12 2 - - 203
-
Marcolungo, Luca; Passera, Alessandro; Maestri, Simone; Segala, Elena; Alfano,
Massimiliano; Gaffuri, Francesca; Marturano, Giovanni; Casati, Paola; Bianco,
Piero; Delledonne, Massimo Real-Time On-Site Diagnosis of Quarantine Pathogens
in Plant Tissues by Nanopore-Based Sequencing Pathogens EN Article plant
pathogen; diagnostics; subspecies; nanopore sequencing; MinION Rapid and
sensitive assays for the identification of plant pathogens are necessary for the
effective management of crop diseases. The main limitation of current diagnostic
testing is the inability to combine broad and sensitive pathogen detection with the
identification of key strains, pathovars, and subspecies. Such discrimination is
necessary for quarantine pathogens, whose management is strictly dependent on
genotype identification. To address these needs, we have established and evaluated
a novel all-in-one diagnostic assay based on nanopore sequencing for the detection
and simultaneous characterization of quarantine pathogens, using Xylella fastidiosa
as a case study. The assay proved to be at least as sensitive as standard
diagnostic tests and the quantitative results agreed closely with qPCR-based
analysis. The same sequencing results also allowed discrimination between
subspecies when present either individually or in combination. Pathogen detection
and typing were achieved within 13 min of sequencing owing to the use of an
internal control that allowed to stop sequencing when sufficient data had
accumulated. These advantages, combined with the use of portable equipment, will
facilitate the development of next-generation diagnostic assays for the efficient
monitoring of other plant pathogens. Department of Biotechnology, University
of Verona, Strada Le Grazie 15, 37134 Verona, Italy luca.marcolungo@univr.it;
alessandro.passera@unimi.it; simone.maestri@univr.it; elena.segala@univr.it;
massimiliano.alfano@univr.it; francesca_gaffuri_cnt@regione.lombardia.it;
giovanni.marturano@univr.it; paola.casati@unimi.it; piero.bianco@unimi.it;
massimo.delledonne@univr.it 10.3390/pathogens11020199 2022 11 2 -
- 199 -
Willenbücher, Katharina; Wibberg, Daniel; Huang, Liren; Conrady, Marius; Ramm,
Patrice; Gätcke, Julia; Busche, Tobias; Brandt, Christian; Szewzyk, Ulrich;
Schlüter, Andreas; Barrero Canosa, Jimena; Maus, Irena Phage Genome Diversity
in a Biogas-Producing Microbiome Analyzed by Illumina and Nanopore GridION
Sequencing Microorganisms EN Article virome; phage particle extraction
protocol; phage enrichment; virome structure; bacteriophages; fragment recruitment
The microbial biogas network is complex and intertwined, and therefore
relatively stable in its overall functionality. However, if key functional groups
of microorganisms are affected by biotic or abiotic factors, the entire efficacy
may be impaired. Bacteriophages are hypothesized to alter the steering process of
the microbial network. In this study, an enriched fraction of virus-like particles
was extracted from a mesophilic biogas reactor and sequenced on the Illumina MiSeq
and Nanopore GridION sequencing platforms. Metagenome data analysis resulted in
identifying 375 metagenome-assembled viral genomes (MAVGs). Two-thirds of the
classified sequences were only assigned to the superkingdom Viruses and the
remaining third to the family Siphoviridae, followed by Myoviridae, Podoviridae,
Tectiviridae, and Inoviridae. The metavirome showed a close relationship to the
phage genomes that infect members of the classes Clostridia and Bacilli. Using
publicly available biogas metagenomic data, a fragment recruitment approach showed
the widespread distribution of the MAVGs studied in other biogas microbiomes. In
particular, phage sequences from mesophilic microbiomes were highly similar to the
phage sequences of this study. Accordingly, the virus particle enrichment approach
and metavirome sequencing provided additional genome sequence information for novel
virome members, thus expanding the current knowledge of viral genetic diversity in
biogas reactors. System Microbiology, Department Bioengineering, Leibniz Institute
for Agricultural Engineering and Bioeconomy (ATB), Max-Eyth-Allee 100, 14469
Potsdam, Germany kwillenbuecher@atb-potsdam.de; dwibberg@cebitec.uni-bielefeld.de;
huanglr@cebitec.uni-bielefeld.de; marius.conrady@iasp.hu-berlin.de;
patrice.ramm@iasp.hu-berlin.de; julia.gaetcke@hu-berlin.de; tbusche@cebitec.uni-
bielefeld.de; christian.brandt@med.uni-jena.de; ulrich.szewzyk@tu-berlin.de;
aschluet@cebitec.uni-bielefeld.de; jimena.barrerocanosa@tu-berlin.de;
irena.maus@cebitec.uni-bielefeld.de 10.3390/microorganisms10020368 2022 10
2 - - 368 -
Glushkevich, Anna; Spechenkova, Nadezhda; Fesenko, Igor; Knyazev, Andrey;
Samarskaya, Viktoriya; Kalinina, Natalia; Taliansky, Michael; Love, Andrew
Transcriptomic Reprogramming, Alternative Splicing and RNA Methylation in
Potato (Solanum tuberosum L.) Plants in Response to Potato Virus Y Infection Plants
EN Article potato virus Y; heat stress; complex stress; direct RNA-
seq; lncRNAs; PARylation Plant-virus interactions are greatly influenced by
environmental factors such as temperatures. In virus-infected plants, enhanced
temperature is frequently associated with more severe symptoms and higher virus
content. However, the mechanisms involved in controlling the temperature regulation
of plant-virus interactions are poorly characterised. To elucidate these further,
we analysed the responses of potato plants cv Chicago to infection by potato virus
Y (PVY) at normal (22 °C) and elevated temperature (28 °C), the latter of
which is known to significantly increase plant susceptibility to PVY. Using RNAseq
analysis, we showed that single and combined PVY and heat-stress treatments caused
dramatic changes in gene expression, affecting the transcription of both protein-
coding and non-coding RNAs. Among the newly identified genes responsive to PVY
infection, we found genes encoding enzymes involved in the catalysis of polyamine
formation and poly ADP-ribosylation. We also identified a range of novel non-coding
RNAs which were differentially produced in response to single or combined PVY and
heat stress, that consisted of antisense RNAs and RNAs with miRNA binding sites.
Finally, to gain more insights into the potential role of alternative splicing and
epitranscriptomic RNA methylation during combined stress conditions, direct RNA
nanopore sequencing was performed. Our findings offer insights for future studies
of functional links between virus infections and transcriptome reprogramming, RNA
methylation and alternative splicing. Shemyakin-Ovchinnikov Institute of
Bioorganic Chemistry of the Russian Academy of Sciences, 117997 Moscow, Russia
zverochek2@gmail.com; rysalka47@gmail.com; fesigor@gmail.com;
agrofak@gmail.com; viktoriya.samarskaya2012@yandex.ru; kalinina@belozersky.msu.ru;
michael.taliansky@hutton.ac.uk; andrew.love@hutton.ac.uk 10.3390/plants11050635
2022 11 5 - - 635 -
Petrone, Joseph; Muñoz-Beristain, Alam; Glusberger, Paula; Russell, Jordan;
Triplett, Eric Unamplified, Long-Read Metagenomic Sequencing Approach to Close
Endosymbiont Genomes of Low-Biomass Insect Populations Microorganisms EN
Article psyllid; insect metagenome; next-generation sequencing; Oxford
Nanopore; PacBio; low-biomass; unamplified; genomics; long-read assembly With
the current advancements in DNA sequencing technology, the limiting factor in long-
read metagenomic assemblies is now the quantity and quality of input DNA. Although
these requirements can be met through the use of axenic bacterial cultures or large
amounts of biological material, insect systems that contain unculturable bacteria
or that contain a low amount of available DNA cannot fully utilize the benefits of
third-generation sequencing. The citrus greening disease insect vector Diaphorina
citri is an example that exhibits both of these limitations. Although endosymbiont
genomes have mostly been closed after the short-read sequencing of amplified
template DNA, creating de novo long-read genomes from the unamplified DNA of an
insect population may benefit communities using bioinformatics to study insect
pathosystems. Here all four genomes of the infected D. citri microbiome were
sequenced to closure using unamplified template DNA and two long-read sequencing
technologies. Avoiding amplification bias and using long reads to assemble the
bacterial genomes allowed for the circularization of the Wolbachia endosymbiont of
Diaphorina citri for the first time and paralleled the annotation context of all
four reference genomes without utilizing a traditional hybrid assembly. The
strategies detailed here are suitable for the sequencing of other insect systems
for which the input DNA, time, and cost are an issue. Microbiology and Cell Science
Department, Institute of Food and Agricultural Sciences, University of Florida,
Gainesville, FL 32603, USA josephpetrone@ufl.edu; amunozberistain@ufl.edu;
priosglus@ufl.edu; russell.j.7@ufl.edu; ewt@ufl.edu
10.3390/microorganisms10030513 2022 10 3 - - 513 -
Tamizi, Amin-Asyraf; Mat-Amin, Noriha; Weaver, Jack; Olumakaiye, Richard; Akbar,
Muhamad; Jin, Sophie; Bunawan, Hamidun; Alberti, Fabrizio Genome Sequencing and
Analysis of Trichoderma (Hypocreaceae) Isolates Exhibiting Antagonistic Activity
against the Papaya Dieback Pathogen, Erwinia mallotivora Journal of Fungi EN
Article biocontrol; fungus; Trichoderma; Gram-negative; Erwinia
Erwinia mallotivora, the causal agent of papaya dieback disease, is a
devastating pathogen that has caused a tremendous decrease in Malaysian papaya
export and affected papaya crops in neighbouring countries. A few studies on
bacterial species capable of suppressing E. mallotivora have been reported, but the
availability of antagonistic fungi remains unknown. In this study, mycelial
suspensions from five rhizospheric Trichoderma isolates of Malaysian origin were
found to exhibit notable antagonisms against E. mallotivora during co-cultivation.
We further characterised three isolates, Trichoderma koningiopsis UKM-M-UW RA5,
UKM-M-UW RA6, and UKM-M-UW RA3a, that showed significant growth inhibition zones on
plate-based inhibition assays. A study of the genomes of the three strains through
a combination of Oxford nanopore and Illumina sequencing technologies highlighted
potential secondary metabolite pathways that might underpin their antimicrobial
properties. Based on these findings, the fungal isolates are proven to be useful as
potential biological control agents against E. mallotivora, and the genomic data
opens possibilities to further explore the underlying molecular mechanisms behind
their antimicrobial activity, with potential synthetic biology applications. Agri-
Omics and Bioinformatics Programme, Biotechnology and Nanotechnology Research
Centre, Malaysian Agricultural Research and Development Institute Headquarters
(MARDI), Serdang 43400, Selangor, Malaysia aminasyraf@mardi.gov.my;
noriha@mardi.gov.my; jack.weaver@warwick.ac.uk; richard.olumakaiye@warwick.ac.uk;
muhdafiq.akbar@gmail.com; sophie.jin@warwick.ac.uk; hamidun.bunawan@ukm.edu.my;
f.alberti@warwick.ac.uk 10.3390/jof8030246 2022 8 3 - - 246
-
Rutjens, Sofie; Vereecke, Nick; De Spiegelaere, Ward; Croubels, Siska; Devreese,
Mathias Intestinal Exposure to Ceftiofur and Cefquinome after Intramuscular
Treatment and the Impact of Ceftiofur on the Pig Fecal Microbiome and Resistome
Antibiotics EN Article intramuscular administration; cephalosporins;
UHPLC-MS/MS; gut and fecal excretion; swine; fecal microbiome; resistome
Optimization of antimicrobial treatment during a bacterial infection in
livestock requires in-depth knowledge of the impact of antimicrobial therapy on the
pathogen and commensal microbiota. Once administered antimicrobials and/or their
metabolites are excreted either by the kidneys through urine and/or by the
intestinal tract through feces, causing antimicrobial pressure and possibly the
emergence of resistance in the gastro-intestinal tract. So far, the excretion of
ceftiofur and cefquinome in the intestinal tract of pigs has not been described.
The objective of this study was to investigate the excretion of ceftiofur and
cefquinome in the different segments of the gut and feces after intramuscular
administration. Therefore, 16 pigs were treated either with ceftiofur (n = 8) or
cefquinome (n = 8), and feces were collected during the entire treatment period.
The presence of ceftiofur and desfuroylceftiofuracetamide or cefquinome were
quantified via liquid chromatography–tandem mass spectrometry. At the end of
the treatment, pigs were euthanized, and samples from the duodenum, jejunum, ileum
and cecum were analyzed. In feces, no active antimicrobial residues could be
measured, except for one ceftiofur-treated pig. In the gut segments, the
concentration of both antimicrobials increased from duodenum toward the ileum, with
a maximum in the ileum (187.8 ± 101.7 ng·g−1 ceftiofur-related
residues, 57.8 ± 37.5 ng·g−1 cefquinome) and sharply decreased
in the cecum (below the limit of quantification for ceftiofur-related residues, 6.4
± 4.2 ng·g−1 cefquinome). Additionally, long-read Nanopore
sequencing and targeted quantitative polymerase chain reaction (qPCR) were
performed in an attempt to clarify the discrepancy in fecal excretion of ceftiofur-
related residues between pigs. In general, there was an increase in Prevotella,
Bacteroides and Faecalibacterium and a decrease in Escherichia and Clostridium
after ceftiofur administration (q-value < 0.05). The sequencing and qPCR could
not provide an explanation for the unexpected excretion of ceftiofur-related
residues in one pig out of eight. Overall, this study provides valuable information
on the gut excretion of parenteral administered ceftiofur and cefquinome.
Department of Pathobiology, Pharmacology and Zoological Medicine, Faculty of
Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium
sofie.rutjens@ugent.be; nick.vereecke@pathosense.com;
ward.despiegelaere@ugent.be; siska.croubels@ugent.be; mathias.devreese@ugent.be
 10.3390/antibiotics11030342 2022 11 3 - - 342 -
Chang, Weizhong; Jiao, Xiaoli; Sui, Hongyan; Goswami, Suranjana; Sherman, Brad;
Fromont, Caroline; Caravaca, Juan; Tran, Bao; Imamichi, Tomozumi Complete Genome
Sequence of Herpes Simplex Virus 2 Strain G Viruses EN Article HSV-2;
complete genome sequence; ‘α’ sequence; packaging signals; genome termini; strain G
Herpes simplex virus type 2 (HSV-2) is a common causative agent of genital
tract infections. Moreover, HSV-2 and HIV infection can mutually increase the risk
of acquiring another virus infection. Due to the high GC content and highly
repetitive regions in HSV-2 genomes, only the genomes of four strains have been
completely sequenced (HG52, 333, SD90e, and MS). Strain G is commonly used for HSV-
2 research, but only a partial genome sequence has been assembled with Illumina
sequencing reads. In the current study, we de novo assembled and annotated the
complete genome of strain G using PacBio long sequencing reads, which can span the
repetitive regions, analyzed the ‘α’ sequence, which plays key
roles in HSV-2 genome circulation, replication, cleavage, and packaging of progeny
viral DNA, identified the packaging signals homologous to HSV-1 within the
‘α’ sequence, and determined both termini of the linear genome
and cleavage site for the process of concatemeric HSV-2 DNA produced via rolling-
circle replication. In addition, using Oxford Nanopore Technology sequencing reads,
we visualized four HSV-2 genome isomers at the nucleotide level for the first time.
Furthermore, the coding sequences of HSV-2 strain G have been compared with those
of HG52, 333, and MS. Moreover, phylogenetic analysis of strain G and other diverse
HSV-2 strains has been conducted to determine their evolutionary relationship. The
results will aid clinical research and treatment development of HSV-2. Laboratory
of Human Retrovirology and lmmunoinformatics, Frederick National Laboratory for
Cancer Research, Frederick, MD 21702, USA weizhong.chang@nih.gov;
jiaoxl@protonmail.com; hongyan.sui@nih.gov; suranjana.goswami@nih.gov;
bsherman@mail.nih.gov; caroline.fromont@nih.gov; juanmanuel.carava@nih.gov;
tranb2@mail.nih.gov; timamichi@mail.nih.gov 10.3390/v14030536 2022 14 3
- - 536 -
Ullmann, Lena; Wibberg, Daniel; Busche, Tobias; Rückert, Christian; Müsgens,
Andreas; Kalinowski, Jörn; Blank, Lars Seventeen Ustilaginaceae High-Quality
Genome Sequences Allow Phylogenomic Analysis and Provide Insights into Secondary
Metabolite Synthesis Journal of Fungi EN Article AAI; ANI; POCP; Oxford
nanopore; phylogenomics; Ustilaginaceae; metabolic engineering; itaconic acid;
ustilagic acid; smut fungi; Ustilago maydis The family of Ustilaginaceae
belongs to the order of Basidiomycetes. Despite their plant pathogenicity causing,
e.g., corn smut disease, they are also known as natural producers of value-added
chemicals such as extracellular glycolipids, organic acids, and polyols. Here, we
present 17 high-quality draft genome sequences (N50 > 1 Mb) combining third-
generation nanopore and second-generation Illumina sequencing. The data were
analyzed with taxonomical genome-based bioinformatics methods such as Percentage of
Conserved Proteins (POCP), Average Nucleotide Identity (ANI), and Average Amino
Acid Identity (AAI) analyses indicating that a reclassification of the
Ustilaginaceae family might be required. Further, conserved core genes were
determined to calculate a phylogenomic core genome tree of the Ustilaginaceae that
also supported the results of the other phylogenomic analysis. In addition, to
genomic comparisons, secondary metabolite clusters (e.g., itaconic acid,
mannosylerythritol lipids, and ustilagic acid) of biotechnological interest were
analyzed, whereas the sheer number of clusters did not differ much between species.
iAMB—Institute of Applied Microbiology, ABBt—Aachen Biology and
Biotechnology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany
lena.ullmann@rwth-aachen.de; dwibberg@cebitec.uni-bielefeld.de;
tbusche@cebitec.uni-bielefeld.de; cruecker@cebitec.uni-bielefeld.de;
andreas.muesgens@rwth-aachen.de; joern@cebitec.uni-bielefeld.de;
lars.blank@rwth.aachen.de 10.3390/jof8030269 2022 8 3 - -
269 -
Cano, Irene; Parker, Abigail; Ward, Georgia; Green, Matthew; Ross, Stuart; Bignell,
John; Daumich, Caroline; Kerr, Rose; Feist, Stephen; Batista, Frederico First
Detection of Francisella halioticida Infecting a Wild Population of Blue Mussels
Mytilus edulis in the United Kingdom Pathogens EN Article Francisella
halioticida; blue mussels Mytilus edulis; prokaryote cyst; granulocytoma;
intracellular bacterium; Nanopore sequencing; 16S rRNA geneIn the last decade,
declines in the population of wild blue mussels Mytilus edulis in the Tamar estuary
(United Kingdom) have been noted. In archived samples collected from 2013 to 2019,
between 7% (in 2013) and 18% (in 2019) showed large granulocytoma and haemocytic
infiltration in the interstitial tissue of the digestive gland. Four samples were
selected for 16S rRNA gene Nanopore sequencing. A consensus sequence of 1449 bp
showed nucleotide similarities between 99.93–100% with published sequences of
Francisella halioticida. In situ hybridisation (ISH) confirmed the presence of F.
halioticida DNA within individual granulocytes of granulocytomas and also in
prokaryotic-like inclusion bodies within the digestive epithelial cells. The design
of diagnostic tests for surveillance of F. halioticida, including more specific ISH
probes and sequencing the genome of the isolates infecting mussels, will shed more
light on the pathogenicity and spread of this pathogen. Cefas Weymouth
Laboratory, International Centre of Excellence for Aquatic Animal Health, Barrack
Road, Weymouth DT4 8UB, UK irene.canocejas@cefas.co.uk;
abigail.parker@cefas.co.uk; georgia.m.ward@cefas.co.uk; matthew.green@cefas.co.uk;
stuart.ross@cefas.co.uk; john.bignell@cefas.co.uk; caroline.daumich@cefas.co.uk;
rose.kerr@cefas.co.uk; oie.cceaad@cefas.co.uk; frederico.batista@cefas.co.uk
10.3390/pathogens11030329 2022 11 3 - - 329 -
Bianco, Luca; Moser, Mirko; Silverj, Andrea; Micheletti, Diego; Lorenzin, Giovanni;
Collini, Lucia; Barbareschi, Mattia; Lanzafame, Paolo; Segata, Nicola; Pindo,
Massimo; Franceschi, Pietro; Rota-Stabelli, Omar; Rizzoli, Annapaola; Fontana,
Paolo; Donati, Claudio On the Origin and Propagation of the COVID-19 Outbreak in
the Italian Province of Trento, a Tourist Region of Northern Italy Viruses
EN Article SARS-CoV-2; genome; transmission Background: Trentino is
an Italian province with a tourism-based economy, bordering the regions of Lombardy
and Veneto, where the two earliest and largest outbreaks of COVID-19 occurred in
Italy. The earliest cases in Trentino were reported in the first week of March
2020, with most of the cases occurring in the winter sport areas in the Dolomites
mountain range. The number of reported cases decreased over the summer months and
was followed by a second wave in the autumn and winter of 2020. Methods: we
performed high-coverage Oxford Nanopore sequencing of 253 positive SARS-CoV-2 swabs
collected in Trentino between March and December 2020. Results: in this work, we
analyzed genome sequences to trace the routes through which the virus entered the
area, and assessed whether the autumnal resurgence could be attributed to lineages
persisting undetected during summer, or as a consequence of new introductions.
Conclusions: Comparing the draft genomes analyzed with a large selection of
European sequences retrieved from GISAID we found that multiple introductions of
the virus occurred at the early stage of the epidemics; the two epidemic waves were
unrelated; the second wave was due to reintroductions of the virus in summer when
traveling restrictions were uplifted. Research and Innovation Centre,
Fondazione Edmund Mach, Via Mach 1, 38098 San Michele all’Adige, Italy
luca.bianco@fmach.it; mirko.moser@fmach.it; andrea.silverj@unitn.it;
diego.micheletti@fmach.it; giovanni.lorenzin@apss.tn.it; lucia.collini@apss.tn.it;
mattia.barbareschi@apss.tn.it; paolo.lanzafame@apss.tn.it; nicola.segata@unitn.it;
massimo.pindo@fmach.it; pietro.franceschi@fmach.it; omar.rota@fmach.it;
annapaola.rizzoli@fmach.it; paolo.fontana@fmach.it; claudio.donati@fmach.it
10.3390/v14030580 2022 14 3 - - 580 -
Craddock, Hillary; Motro, Yair; Zilberman, Bar; Khalfin, Boris; Bardenstein,
Svetlana; Moran-Gilad, Jacob Long-Read Sequencing and Hybrid Assembly for Genomic
Analysis of Clinical Brucella melitensis Isolates Microorganisms EN
Article brucellosis; whole-genome sequencing; clinical genomics
Brucella melitensis is a key etiological agent of brucellosis and has been
increasingly subject to characterization using sequencing methodologies. This study
aimed to investigate and compare short-read, long-read, and hybrid assemblies of B.
melitensis. Eighteen B. melitensis isolates from Southern Israel were sequenced
using Illumina and the Oxford Nanopore (ONP) MinION, and hybrid assemblies were
generated with ONP long reads scaffolded on Illumina short reads. Short reads were
assembled with INNUca with SPADes, long reads and hybrid with dragonflye. Abricate
with the virulence factor database (VFDB) and in silico PCR (for the genes BetB,
BPE275, BSPB, manA, mviN, omp19, perA, PrpA, VceC, and ureI) were used for
identifying virulence genes, and a total of 61 virulence genes were identified in
short-read, long-read, and hybrid assemblies of all 18 isolates. The phylogenetic
analysis using long-read assemblies revealed several inconsistencies in cluster
assignment as compared to using hybrid and short-read assemblies. Overall, hybrid
assembly provided the most comprehensive data, and stand-alone short-read
sequencing provided comparable data to stand-alone long-read sequencing regarding
virulence genes. For genomic epidemiology studies, stand-alone ONP sequencing may
require further refinement in order to be useful in endemic settings.
Microbiology, Advanced Genomics and Infection Control Application Laboratory
(MAGICAL) Group, Department of Health Systems Management, School of Public Health,
Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105,
Israel hcraddock5@gmail.com; motroy@post.bgu.ac.il; barsch@post.bgu.ac.il;
boriskh83@gmail.com; svetab@moag.gov.il; giladko@post.bgu.ac.il
10.3390/microorganisms10030619 2022 10 3 - - 619 -
Kong, Ping; Li, Xiaoping; Gouker, Fred; Hong, Chuanxue cDNA Transcriptome of
Arabidopsis Reveals Various Defense Priming Induced by a Broad-Spectrum Biocontrol
Agent Burkholderia sp. SSG International Journal of Molecular Sciences EN
Article Arabidopsis; cDNA transcriptome; Oxford Nanopore Technology (ONT)
sequencing; defense priming; induced systemic resistance (ISR); systemic acquired
resistance (SAR); biocontrol agent; leaf endophyte; Burkholderia sp. Burkholderia
sp. SSG is a potent biological control agent. Even though its survival on the leaf
surface declined rapidly, SSG provided extended, moderate plant protection from a
broad spectrum of pathogens. This study used Arabidopsis Col-0 and its mutants,
eds16-1, npr1-1, and pad4-1 as model plants and compared treated plants with non-
treated controls to elucidate whether SSG triggers plant defense priming. Only
eds16-1 leaves with SSG became purplish, suggesting the involvement of salicylic
acid (SA) in SSG-induced priming. cDNA sequencing of Col-0 plants and differential
gene expression analysis identified 120 and 119 differentially expressed genes
(DEGs) at 6- and 24-h post-treatment (hpt) with SSG, respectively. Most of these
DEGs encoded responses to biotic and abiotic stimuli or stresses; four DEGs had
more than two isoforms. A total of 23 DEGs were shared at 6 and 24 hpt, showing
four regulation patterns. Functional categorization of these shared DEGs, and 44
very significantly upregulated DEGs revealed that SSG triggered various defense
priming mechanisms, including responses to phosphate or iron deficiency, modulation
of defense-linked SA, jasmonic acid, ethylene, and abscisic acid pathways, defense-
related gene regulation, and chromatin modification. These data support that SSG is
an induced systemic resistance (ISR) trigger conferring plant protection upon
pathogen encounter. Virginia Tech, Hampton Roads Agricultural Research and
Extension Center, 1444 Diamond Springs Road, Virginia Beach, VA 23455, USA
pkong@vt.edu; lixiaopi@vt.edu; fred.gouker@usda.gov; chhong2@vt.edu
10.3390/ijms23063151 2022 23 6 - - 3151 -
Conde, Daniel; Garrido, Pablo; Calvelo, Martín; Piñeiro, Ángel; Garcia-Fandino,
Rebeca Molecular Dynamics Simulations of Transmembrane Cyclic Peptide
Nanotubes Using Classical Force Fields, Hydrogen Mass Repartitioning, and Hydrogen
Isotope Exchange Methods: A Critical Comparison International Journal of Molecular
Sciences EN Article cyclic peptides; antimicrobial peptides; self-
assembled peptide nanotubes; molecular dynamics; force field; hydrogen mass
repartitioning; hydrogen isotope exchange Self-assembled cyclic peptide nanotubes
with alternating D- and L-amino acid residues in the sequence of each subunit have
attracted a great deal of attention due to their potential for new nanotechnology
and biomedical applications, mainly in the field of antimicrobial peptides.
Molecular dynamics simulations can be used to characterize these systems with
atomic resolution at different time scales, providing information that is difficult
to obtain via wet lab experiments. However, the performance of classical force
fields typically employed in the simulation of biomolecules has not yet been
extensively tested with this kind of highly constrained peptide. Four different
classical force fields (AMBER, CHARMM, OPLS, and GROMOS), using a nanotube formed
by eight D,L-α-cyclic peptides inserted into a lipid bilayer as a model
system, were employed here to fill this gap. Significant differences in the pseudo-
cylindrical cavities formed by the nanotubes were observed, the most important
being the diameter of the nanopores, the number and location of confined water
molecules, and the density distribution of the solvent molecules. Furthermore,
several modifications were performed on GROMOS54a7, aiming to explore acceleration
strategies of the MD simulations. The hydrogen mass repartitioning (HMR) and
hydrogen isotope exchange (HIE) methods were tested to slow down the fastest
degrees of freedom. These approaches allowed a significant increase in the time
step employed in the equation of the motion integration algorithm, from 2 fs up to
5–7 fs, with no serious changes in the structural and dynamical properties of
the nanopores. Subtle differences with respect to the simulations with the
unmodified force fields were observed in the concerted movements of the cyclic
peptides, as well as in the lifetime of several H-bonds. All together, these
results are expected to contribute to better understanding of the behavior of self-
assembled cyclic peptide nanotubes, as well as to support the methods tested to
speed up general MD simulations; additionally, they do provide a number of
quantitative descriptors that are expected to be used as a reference to design new
experiments intended to validate and complement computational studies of
antimicrobial cyclic peptides. Center for Research in Biological Chemistry and
Molecular Materials, Departamento de Química Orgánica, Universidade de Santiago de
Compostela, Campus Vida s/n, 15782 Santiago de Compostela, Spain
daniel.conde@rai.usc.es; pablo.fernandez@usc.es; martin.calvelo.souto@usc.es;
angel.pineiro@usc.es; rebeca.garcia.fandino@usc.es 10.3390/ijms23063158 2022
23 6 - - 3158 -
Sajnaga, Ewa; Skowronek, Marcin; Kalwasińska, Agnieszka; Kazimierczak, Waldemar;
Lis, Magdalena; Jach, Monika; Wiater, Adrian Comparative Nanopore Sequencing-
Based Evaluation of the Midgut Microbiota of the Summer Chafer (Amphimallon
solstitiale L.) Associated with Possible Resistance to Entomopathogenic Nematodes
International Journal of Environmental Research and Public Health EN
Article gut microbiota; entomopathogenic nematodes; pathogen resistance;
host–pathogen interactions; Scarabaeidae; nanopore sequencing; metataxonomics; pest
biocontrol Root-feeding Amphimallon solstitiale larvae and certain other scarab
beetles are the main soil-dwelling pests found in Europe, while entomopathogenic
nematodes (EPN) have been used as a biocontrol agent against these species. Our
study provides the first detailed characterization of the bacterial community of
the midgut in wild A. solstitiale larvae, based on the nanopore sequencing of the
16S rRNA gene. In the whole dataset, we detected 2586 different genera and 11,641
species, with only 83 diverse bacterial genera shared by all studied individuals,
which may represent members of the core midgut microbiota of A. solstitiale larvae.
Subsequently, we compared the midgut microbiota of EPN-resistant and T0 (prior to
EPN exposure) individuals, hypothesizing that resistance to this parasitic
infection may be linked to the altered gut community. Compared to the control, the
resistant insect microbiota demonstrated lower Shannon and Evenness indices and
significant differences in the community structure. Our studies confirmed that the
gut microbiota alternation is associated with resistant insects; however, there are
many processes involved that can affect the bacterial community. Further research
on the role of gut microbiota in insect-parasitic nematode interaction may
ultimately lead to the improvement of biological control strategies in insect pest
management. Laboratory of Biocontrol, Production, and Application of EPN, Centre
for Interdisciplinary Research, The John Paul II Catholic University of Lublin,
Konstantynów 1J, 20-708 Lublin, Poland ewa.sajnaga@kul.pl;
marcin.skowronek@kul.pl; kala@umk.pl; waldemar.kazimierczak@kul.pl;
magdalena.lis@kul.pl; monika.jach@kul.pl; adrian.wiater@mail.umcs.pl
10.3390/ijerph19063480 2022 19 6 - - 3480 -
Napieralski, Adam; Nowak, Robert Basecalling Using Joint Raw and Event Nanopore
Data Sequence-to-Sequence Processing Sensors EN Article basecalling;
sequence-to-sequence; nanopore; bioinformatics; encoder decoder; attention; neural
network; machine learning; joint processing Third-generation DNA sequencers
provided by Oxford Nanopore Technologies (ONT) produce a series of samples of an
electrical current in the nanopore. Such a time series is used to detect the
sequence of nucleotides. The task of translation of current values into nucleotide
symbols is called basecalling. Various solutions for basecalling have already been
proposed. The earlier ones were based on Hidden Markov Models, but the best ones
use neural networks or other machine learning models. Unfortunately, achieved
accuracy scores are still lower than competitive sequencing techniques, like
Illumina’s. Basecallers differ in the input data type—currently, most
of them work on a raw data straight from the sequencer (time series of current).
Still, the approach of using event data is also explored. Event data is obtained by
preprocessing of raw data and dividing it into segments described by several
features computed from raw data values within each segment. We propose a novel
basecaller that uses joint processing of raw and event data. We define basecalling
as a sequence-to-sequence translation, and we use a machine learning model based on
an encoder–decoder architecture of recurrent neural networks. Our model
incorporates twin encoders and an attention mechanism. We tested our solution on
simulated and real datasets. We compare the full model accuracy results with its
components: processing only raw or event data. We compare our solution with the
existing ONT basecaller—Guppy. Results of numerical experiments show that
joint raw and event data processing provides better basecalling accuracy than
processing each data type separately. We implement an application called Ravvent,
freely available under MIT licence. Institute of Computer Science, Faculty of
Electronics and Information Technology, Warsaw University of Technology, 00-665
Warsaw, Poland adam.napieralski.stud@pw.edu.pl; robert.nowak@pw.edu.pl
10.3390/s22062275 2022 22 6 - - 2275 -
Liao, Yu-Chieh; Chen, Feng-Jui; Chuang, Min-Chieh; Wu, Han-Chieh; Ji, Wan-Chen; Yu,
Guann-Yi; Huang, Tsi-Shu High-Integrity Sequencing of Spike Gene for SARS-CoV-
2 Variant Determination International Journal of Molecular Sciences EN
Article SARS-CoV-2; variant; spike gene; nanopore sequencing For tiling
of the SARS-CoV-2 genome, the ARTIC Network provided a V4 protocol using 99 pairs
of primers for amplicon production and is currently the widely used amplicon-based
approach. However, this technique has regions of low sequence coverage and is
labour-, time-, and cost-intensive. Moreover, it requires 14 pairs of primers in
two separate PCRs to obtain spike gene sequences. To overcome these disadvantages,
we proposed a single PCR to efficiently detect spike gene mutations. We proposed a
bioinformatic protocol that can process FASTQ reads into spike gene consensus
sequences to accurately call spike protein variants from sequenced samples or to
fairly express the cases of missing amplicons. We evaluated the in silico detection
rate of primer sets that yield amplicon sizes of 400, 1200, and 2500 bp for spike
gene sequencing of SARS-CoV-2 to be 59.49, 76.19, and 92.20%, respectively. The in
silico detection rate of our proposed single PCR primers was 97.07%. We
demonstrated the robustness of our analytical protocol against 3000 Oxford Nanopore
sequencing runs of distinct datasets, thus ensuring high-integrity sequencing of
spike genes for variant SARS-CoV-2 determination. Our protocol works well with the
data yielded from versatile primer designs, making it easy to determine spike
protein variants. Institute of Population Health Sciences, National Health Research
Institutes, No. 35, Keyan Road, Zhunan Town, Miaoli County 35053, Taiwan
jade@nhri.edu.tw; frchen@nhri.edu.tw; mcchuang@thu.edu.tw;
hanjie@nhri.edu.tw; ss870805ss@gmail.com; guannyiy@nhri.edu.tw;
tshuang@vghks.gov.tw 10.3390/ijms23063257 2022 23 6 - - 3257
-
Schiavone, Antonella; Pugliese, Nicola; Samarelli, Rossella; Cumbo, Cosimo;
Minervini, Crescenzio; Albano, Francesco; Camarda, Antonio Factors Affecting the
Quality of Bacterial Genomes Assemblies by Canu after Nanopore Sequencing
Applied Sciences EN Article flongle; MinION; bacterial genome;
Salmonella enterica; plasticity; de novo assembly; Canu; options; quality; contigs
 Long-read sequencing (LRS), like Oxford Nanopore Technologies, is usually
associated with higher error rates compared to previous generations. Factors
affecting the assembly quality are the integrity of DNA, the flowcell efficiency,
and, not least all, the raw data processing. Among LRS-intended de novo assemblers,
Canu is highly flexible, with its dozens of adjustable parameters. Different Canu
parameters were compared for assembling reads of Salmonellaenterica ser.
Bovismorbificans (genome size of 4.8 Mbp) from three runs on MinION (N50 651, 805,
and 5573). Two of them, with low quality and highly fragmented DNA, were not usable
alone for assembly, while they were successfully assembled when combining the reads
from all experiments. The best results were obtained by modifying Canu parameters
related to the error correction, such as corErrorRate (exclusion of overlaps above
a set error rate, set up at 0.40), corMhapSensitivity (the coarse sensitivity
level, set to “high”), corMinCoverage (set to 0 to correct all reads,
regardless the overlaps length), and corOutCoverage (corrects the longest reads up
to the imposed coverage, set to 100). This setting produced two contigs
corresponding to the complete sequences of the chromosome and a plasmid. The
overall results highlight the importance of a tailored bioinformatic analysis.
Department of Veterinary Medicine, University of Bari, 70010 Valenzano, Italy
antonella.schiavone@uniba.it; nicola.pugliese@uniba.it;
rossella.samarelli@uniba.it; cosimo.cumbo@gmail.com; eziominervini@gmail.com;
francesco.albano@uniba.it; antonio.camarda@uniba.it 10.3390/app12063110 2022
12 6 - - 3110 -
Hasanau, Tsimur; Pisarev, Eduard; Kisil, Olga; Nonoguchi, Naosuke; Le Calvez-Kelm,
Florence; Zvereva, Maria Detection of TERT Promoter Mutations as a Prognostic
Biomarker in Gliomas: Methodology, Prospects, and Advances Biomedicines EN
Review telomerase reverse transcriptase; telomerase activation; TERT;
TERT promoter region; TERT mutations; glioma; central nervous system tumors;
molecular biomarkers; noninvasive detection; dPCR; ddPCR; Sanger sequencing; NGS;
MRI This article reviews the existing approaches to determining the TERT promoter
mutational status in patients with various tumoral diseases of the central nervous
system. The operational characteristics of the most common methods and their
transferability in medical practice for the selection or monitoring of personalized
treatments based on the TERT status and other related molecular biomarkers in
patients with the most common tumors, such as glioblastoma, oligodendroglioma, and
astrocytoma, are compared. The inclusion of new molecular markers in the course of
CNS clinical management requires their rapid and reliable assessment. Availability
of molecular evaluation of gliomas facilitates timely decisions regarding patient
follow-up with the selection of the most appropriate treatment protocols.
Significant progress in the inclusion of molecular biomarkers for their subsequent
clinical application has been made since 2016 when the WHO CNS classification first
used molecular markers to classify gliomas. In this review, we consider the
methodological approaches used to determine mutations in the promoter region of the
TERT gene in tumors of the central nervous system. In addition to classical
molecular genetical methods, other methods for determining TERT mutations based on
mass spectrometry, magnetic resonance imaging, next-generation sequencing, and
nanopore sequencing are reviewed with an assessment of advantages and
disadvantages. Beyond that, noninvasive diagnostic methods based on the
determination of the mutational status of the TERT promoter are discussed.
Department of Chemistry, Lomonosov Moscow State University, 119991 Moscow,
Russia mrtimurgasanov@gmail.com; e.pisarev@fbb.msu.ru; olvv@mail.ru;
naosuke.nonoguchi@ompu.ac.jp; lecalvezf@iarc.fr; mzvereva@chem.msu.ru
10.3390/biomedicines10030728 2022 10 3 - - 728 -
Martín-Hernández, Giselle; Müller, Bettina; Brandt, Christian; Hölzer, Martin;
Viehweger, Adrian; Passoth, Volkmar Near Chromosome-Level Genome Assembly and
Annotation of Rhodotorula babjevae Strains Reveals High Intraspecific Divergence
Journal of Fungi EN Article Rhodotorula babjevae; de novo hybrid
assembly; nanopore sequencing; genome divergence; ploidy The genus Rhodotorula
includes basidiomycetous oleaginous yeast species. Rhodotorula babjevae can produce
compounds of biotechnological interest such as lipids, carotenoids, and
biosurfactants from low value substrates such as lignocellulose hydrolysate. High-
quality genome assemblies are needed to develop genetic tools and to understand
fungal evolution and genetics. Here, we combined short- and long-read sequencing to
resolve the genomes of two R. babjevae strains, CBS 7808 (type strain) and DBVPG
8058, at chromosomal level. Both genomes are 21 Mbp in size and have a GC content
of 68.2%. Allele frequency analysis indicates that both strains are tetraploid. The
genomes consist of a maximum of 21 chromosomes with a size of 0.4 to 2.4 Mbp. In
both assemblies, the mitochondrial genome was recovered in a single contig, that
shared 97% pairwise identity. Pairwise identity between most chromosomes ranges
from 82 to 87%. We also found indications for strain-specific extrachromosomal
endogenous DNA. A total of 7591 and 7481 protein-coding genes were annotated in CBS
7808 and DBVPG 8058, respectively. CBS 7808 accumulated a higher number of tandem
duplications than DBVPG 8058. We identified large translocation events between
putative chromosomes. Genome divergence values between the two strains indicate
that they may belong to different species. Department of Molecular Sciences,
Swedish University of Agricultural Sciences, 75007 Uppsala, Sweden
giselle.martin@slu.se; bettina.muller@slu.se; christian.brandt@med.uni-
jena.de; hoelzerm@rki.de; adrian.viehweger@medizin.uni-leipzig.de;
volkmar.passoth@slu.se 10.3390/jof8040323 2022 8 4 - - 323
-
Lihodeevskiy, Georgiy; Shanina, Elena The Use of Long-Read Sequencing to Study
the Phylogenetic Diversity of the Potato Varieties Plastome of the Ural Selection
Agronomy EN Communication potato; Solanum tuberosum L.; plastome;
long-reads; nanopore sequencing Plastid DNA holds a substantial amount of plant
genetic information, including maternal ancestry information. It helps to uncover
interrelations between a wide variety of tuberous species of the genus Solanum to
search for promising sources of high-yielding potato varieties resistant to bio-
and abiotic stressors. This paper demonstrated the opportunities of de novo
assembly of potato plastid DNA and its phylogenetic and genome type identification
based only on Oxford Nanopore Technologies (ONT) long reads. According to our
results, of 28 potato varieties developed at the Ural Research Institute of
Agriculture, 16 varieties had one of the most primitive W-type plastomes. Ten
varieties’ plastomes belonged to the T-type of cultivated Solanum tuberosum
subsp. tuberosum. The varieties Legenda and 15-27-1 were the closest to the wild
species Solanum chacoense plastome. Using long-sequencing reads, we confirmed the
presence of two isoforms of the plastid genome differing in the orientation of SSC
region. We should note that irrespective of sequencing depth and improvements in
software for working with ONT reads, a correct de novo plastome assembly and its
annotation using only long-reads is impossible. The most problematic regions are
homopolymers longer than 5 bp—they account for all detected indels, leading
to a change in the reading frame or the deletion of entire genes. Ural Federal
Agrarian Research Center, Ural Branch of the Russian Academy of Science, 620142
Ekaterinburg, Russia georglihodey@gmail.com; shanina08@yandex.ru
10.3390/agronomy12040846 2022 12 4 - - 846 -
Gaibani, Paolo; Bussini, Linda; Amadesi, Stefano; Bartoletti, Michele; Bovo,
Federica; Lazzarotto, Tiziana; Viale, Pierluigi; Ambretti, Simone Successful
Treatment of Bloodstream Infection due to a KPC-Producing Klebsiella Pneumoniae
Resistant to Imipenem/Relebactam in a Hematological PatientMicroorganisms EN
Article imipenem/relebactam; meropenem/vaborbactam; cross-resistance; bla
KPC -3 Novel carbapenem-β-lactamase inhibitor combination,
imipenem/relebactam (IMI-REL), has been recently approved for treatment of
infections with limited or no alternative treatment options. In this study, we
described the emergence of the IMI-REL-resistance in a KPC-producing Klebsiella
pneumoniae (KPC-Kp) strain collected from a hematological patient with no evidence
of prior colonization. Interestingly, IMI-REL-resistance was associated with
meropenem/vaborbactam (MER-VAB) cross-resistance but was not associated with cross-
resistance to ceftazidime/avibactam (CAZ-AVI). Although treatment with CAZ-AVI and
gentamicin completely eradicated the infection due KPC-Kp cross-resistance to IMI-
REL and MER-VAB, the patient became colonized subsequently by KPC-Kp strains
susceptible to IMI-REL and MER-VAB. Whole-genome sequencing performed by hybrid
approach using Illumina and Oxford Nanopore platforms demonstrated that all KPC-Kp
strains isolated from hematological patient belonged to the ST512 and were clonally
related. Analysis of antimicrobial and porins genes demonstrated that cross-
resistance to IMI-REL and MER-VAB was associated with increased blaKPC-3 copy
number and truncated OmpK35 and OmpK36 with GD134-135 insertion. Phylogenetic
analysis demonstrated that KPC-Kp cross-resistance to IMI-REL and MER-VAB was
clonally related to a KPC-Kp resistant to IMI-REL as previously described,
demonstrating the spread of this multidrug resistant clone in the hematological
unit. In conclusion, the results presented in this study reported the emergence of
cross-resistance to MER-VAB and IMI-REL in a KPC-Kp strain isolated from a
hematological patient and highlight the potential development and diffusion of new
multidrug resistance traits. Microbiology Unit, IRCCS Azienda Ospedaliero-
Universitaria di Bologna, 40138 Bologna, Italy paolo.gaibani@unibo.it;
linda.bussini@gmail.com; stefano.amadesi@gmail.com; m.bartoletti@unibo.it;
federica.bovo@aosp.bo.it; tiziana.lazzarotto@unibo.it; pierluigi.viale@unibo.it;
simone.ambretti@aosp.bo.it 10.3390/microorganisms10040778 2022 10 4
- - 778 -
Cahyani, Inswasti; Putro, Eko; Ridwanuloh, Asep; Wibowo, Satrio; Hariyatun,
Hariyatun; Syahputra, Gita; Akbariani, Gilang; Utomo, Ahmad; Ilyas, Mohammad;
Loose, Matthew; Kusharyoto, Wien; Susanti, Susanti Genome Profiling of SARS-CoV-
2 in Indonesia, ASEAN and the Neighbouring East Asian Countries: Features,
Challenges and Achievements Viruses EN Article ASEAN; COVID-19; genomic
surveillance; GISAID; nanopore; NICCRAT; SARS-CoV-2; variants of concern; whole-
genome sequencing Whole-genome sequencing (WGS) has played a significant role in
understanding the epidemiology and biology of SARS-CoV-2 virus. Here, we
investigate the use of SARS-CoV-2 WGS in Southeast and East Asian countries as a
genomic surveillance during the COVID-19 pandemic. Nottingham–Indonesia
Collaboration for Clinical Research and Training (NICCRAT) initiative has
facilitated collaboration between the University of Nottingham and a team in the
Research Center for Biotechnology, National Research and Innovation Agency (BRIN),
to carry out a small number of SARS-CoV-2 WGS in Indonesia using Oxford Nanopore
Technology (ONT). Analyses of SARS- CoV-2 genomes deposited on GISAID reveal the
importance of clinical and demographic metadata collection and the importance of
open access and data sharing. Lineage and phylogenetic analyses of two periods
defined by the Delta variant outbreak reveal that: (1) B.1.466.2 variants were the
most predominant in Indonesia before the Delta variant outbreak, having a unique
spike gene mutation N439K at more than 98% frequency, (2) Delta variants AY.23 sub-
lineage took over after June 2021, and (3) the highest rate of virus transmissions
between Indonesia and other countries was through interactions with Singapore and
Japan, two neighbouring countries with a high degree of access and travels to and
from Indonesia. COMGen Division, School of Life Sciences, University of
Nottingham, Nottingham NG7 2UH, UK inswasti.cahyani@nottingham.ac.uk;
eko.wahyu.putro@brin.go.id; asep.muhamad.ridwanuloh@brin.go.id;
satrio@pathgen.co.id; hariyatun@brin.go.id; gita.syahputra@brin.go.id;
gilang@pathgen.co.id; ahmad.utomo@gmail.com; mohammad.ilyas@nottingham.ac.uk;
matt.loose@nottingham.ac.uk; wien.kusharyoto@brin.go.id;
susanti.susanti@nottingham.ac.uk 10.3390/v14040778 2022 14 4 - -
778 -
Zhang, Lvhao; Yang, Tian; Yu, Wangyin; Wang, Xiaojun; Zhou, Xiang; Zhou, Xudong
Genome-Wide Study of Conidiation-Related Genes in the Aphid-Obligate Fungal
Pathogen Conidiobolus obscurus (Entomophthoromycotina) Journal of Fungi EN
Article mycopathogen; fungal genome; fungal virulence; genomic and
transcriptomic profiling; insect pathogenic fungi Fungi in the Entomophthorales
order can cause insect disease and epizootics in nature, contributing to biological
pest control in agriculture and forestry. Most Entomophthorales have narrow host
ranges, limited to the arthropod family level; however, rare genomic information
about host-specific fungi has been reported. Conidiation is crucial for
entomopathogenic fungi to explore insect resources owing to the important roles of
conidia in the infection cycle, such as dispersal, adhesion, germination, and
penetration into the host hemocoel. In this study, we analyzed the whole genome
sequence of the aphid-obligate pathogen Conidiobolus obscurus strain ARSEF 7217
(Entomophthoromycotina), using Nanopore technology from Biomarker Technologies
(Beijing, China). The genome size was 37.6 Mb, and encoded 10,262 predicted genes,
wherein 21.3% genes were putatively associated to the pathogen–host
interaction. In particular, the serine protease repertoire in C. obscurus exhibited
expansions in the trypsin and subtilisin classes, which play vital roles in the
fungus’ pathogenicity. Differentially expressed transcriptomic patterns were
analyzed in three conidiation stages (pre-conidiation, emerging conidiation, and
post-conidiation), and 2915 differentially expressed genes were found to be
associated with the conidiation process. Furthermore, a weighted gene co-expression
network analysis showed that 772 hub genes in conidiation are mainly involved in
insect cuticular component degradation, cell wall/membrane biosynthesis, MAPK
signaling pathway, and transcription regulation. Our findings of the genomic and
transcriptomic features of C. obscurus help reveal the molecular mechanism of the
Entomophthorales pathogenicity, which will contribute to improving fungal
applications in pest control. State Key Laboratory of Subtropical Silviculture,
School of Forestry and Biotechnology, Zhejiang A&F University, Hangzhou 311300,
China 2020102081016@stu.zafuedu.cn; 2021102032004@stu.zafu.edu.cn;
yuwangyin98@gmail.com; 2020102082013@stu.zafuedu.cn; xzhou@zafu.edu.cn;
xudong.zhou@zafu.edu.cn 10.3390/jof8040389 2022 8 4 - - 389
-
Martinez, Magaly; Nguyen, Phuong-Vi; Su, Maxwell; Cardozo, Fátima; Valenzuela,
Adriana; Franco, Laura; Galeano, María; Rojas, Leticia; Díaz Acosta, Chyntia;
Fernández, Jonás; Ortiz, Joel; del Puerto, Florencia; Mendoza, Laura; Nara, Eva;
Rojas, Alejandra; Waggoner, Jesse SARS-CoV-2 Variants in Paraguay: Detection and
Surveillance with an Economical and Scalable Molecular Protocol Viruses EN
Article SARS-CoV-2; variants; real-time RT-PCR; Paraguay SARS-CoV-2
variant detection relies on resource-intensive whole-genome sequencing methods. We
sought to develop a scalable protocol for variant detection and surveillance in
Paraguay, pairing rRT-PCR for spike mutations with Nanopore sequencing. A total of
201 acute-phase nasopharyngeal samples were included. Samples were positive for the
SARS-CoV-2 N2 target and tested with the Spike SNP assay to detect mutations
associated with the following variants: alpha (501Y), beta/gamma
(417variant/484K/501Y), delta (452R/478K), and lambda (452Q/490S). Spike SNP calls
were confirmed using amplicon (Sanger) sequencing and whole-genome (Nanopore)
sequencing on a subset of samples with confirmed variant lineages. Samples had a
mean N2 Ct of 20.8 (SD 5.6); 198/201 samples (98.5%) tested positive in the Spike
SNP assay. The most common genotype was 417variant/484K/501Y, detected in 102/198
samples (51.5%), which was consistent with the P.1 lineage (gamma variant) in
Paraguay. No mutations (K417 only) were found in 64/198 (32.3%), and K417/484K was
identified in 22/198 (11.1%), consistent with P.2 (zeta). Seven samples (3.5%)
tested positive for 452R without 478K, and one sample with genotype K417/501Y was
confirmed as B.1.1.7 (alpha). The results were confirmed using Sanger sequencing in
181/181 samples, and variant calls were consistent with Nanopore sequencing in
29/29 samples. The Spike SNP assay could improve population-level surveillance for
mutations associated with SARS-CoV-2 variants and inform the judicious use of
sequencing resources. Departamento de Biología Molecular y Biotecnología,
Instituto de Investigaciones en Ciencias de la Salud, Universidad Nacional de
Asunción, San Lorenzo 111241, Paraguay imartinez@iics.una.py; phuong-
vi.thi.nguyen@emory.edu; max.su@emory.edu; fati.cardozo@hotmail.com;
abvalenzuela80@gmail.com; laurafpy@hotmail.com; maruphd@hotmail.com;
letyroj@hotmail.com; chyntiacarolinadiaz@gmail.com; jonasmfb@gmail.com;
joelortizm15@gmail.com; colepuerto@gmail.com; lauramendozatorres@gmail.com;
megunara@hotmail.com; arojass@iics.una.py; jjwaggo@emory.edu 10.3390/v14050873
2022 14 5 - - 873 -
Rudova, Nataliia; Buttler, Jeremy; Kovalenko, Ganna; Sushko, Mykola; Bolotin,
Vitaliy; Muzykina, Larysa; Zinenko, Oleksandr; Stegniy, Borys; Dunaiev, Yurii;
Sytiuk, Mykola; Gerilovych, Anton; Drown, Devin; Bortz, Eric; Solodiankin, Oleksii
Genetic Diversity of Porcine Circovirus 2 in Wild Boar and Domestic Pigs in
Ukraine Viruses EN Article porcine circovirus; PCV2; domestic pig;
wild boar; genotype; phylogenetics; MinION; Ukraine Porcine circovirus type 2
(PCV2) is responsible for a number of porcine circovirus-associated diseases
(PCVAD) that can severely impact domestic pig herds. For a non-enveloped virus with
a small genome (1.7 kb ssDNA), PCV2 is remarkably diverse, with eight genotypes
(a–h). New genotypes of PCV2 can spread through the migration of wild boar,
which are thought to infect domestic pigs and spread further through the domestic
pig trade. Despite a large swine population, the diversity of PCV2 genotypes in
Ukraine has been under-sampled, with few PCV2 genome sequences reported in the past
decade. To gain a deeper understanding of PCV2 genotype diversity in Ukraine,
samples of blood serum were collected from wild boars (n = 107) that were hunted in
Ukraine during the November–December 2012 hunting season. We found 34/107
(31.8%) prevalence of PCV2 by diagnostic PCR. For domestic pigs, liver samples (n =
16) were collected from a commercial market near Kharkiv in 2019, of which 6 out of
16 (37%) samples were positive for PCV2. We sequenced the genotyping locus ORF2, a
gene encoding the PCV2 viral capsid (Cap), for 11 wild boar and six domestic pig
samples in Ukraine using an Oxford Nanopore MinION device. Of 17 samples with
resolved genotypes, the PCV2 genotype b was the most common in wild boar samples
(10 out of 11, 91%), while the domestic pigs were infected with genotypes b and d.
We also detected genotype b/d and b/a co-infections in wild boars and domestic
pigs, respectively, and for the first time in Ukraine we detected genotype f in a
wild boar from Poltava. Building a maximum-likelihood phylogeny, we identified a
sublineage of PCV2 genotype b infections in both wild and domestic swine,
suggesting a possible epizootic cluster and an ecological interaction between wild
boar and domestic pig populations in northeastern Ukraine. National Scientific
Center Institute of Experimental and Clinical Veterinary Medicine, 61023 Kharkiv,
Ukraine rudovanatawa@ukr.net; jdbuttler@alaska.edu; ak2388@cam.ac.uk;
m.i.sushko@gmail.com; vbolotin@hotmail.de; loramuzykina@i.ua;
oleksandrzinenko@gmail.com; boris.stegniy@gmail.com; dunaev2975@gmail.com;
snp1978@ukr.net; antger2011@gmail.com; dmdrown@alaska.edu; ebortz@alaska.edu;
olexii.solod@gmail.com 10.3390/v14050924 2022 14 5 - - 924 -
Zhou, You; Ren, Meishen; Zhang, Pengfei; Jiang, Dike; Yao, Xueping; Luo, Yan; Yang,
Zexiao; Wang, Yin Application of Nanopore Sequencing in the Detection of Foodborne
Microorganisms Nanomaterials EN Review foodborne diseases; nanopore
sequencing technology; WGS; metagenomics; real-time monitoring; poultry health;
COVID-19 Foodborne pathogens have become the subject of intense interest because
of their high incidence and mortality worldwide. In the past few decades, people
have developed many methods to solve this challenge. At present, methods such as
traditional microbial culture methods, nucleic acid or protein-based pathogen
detection methods, and whole-genome analysis are widely used in the detection of
pathogenic microorganisms in food. However, these methods are limited by time-
consuming, cumbersome operations or high costs. The development of nanopore
sequencing technology offers the possibility to address these shortcomings.
Nanopore sequencing, a third-generation technology, has the advantages of simple
operation, high sensitivity, real-time sequencing, and low turnaround time. It can
be widely used in the rapid detection and serotyping of foodborne pathogens. This
review article discusses foodborne diseases, the principle of nanopore sequencing
technology, the application of nanopore sequencing technology in foodborne
pathogens detection, as well as its development prospects. Key Laboratory of Animal
Diseases and Human Health of Sichuan Province, College of Veterinary Medicine,
Sichuan Agricultural University, Chengdu 611130, China
2021203043@stu.sicau.edu.cn; 14773@sicau.edu.cn; 2018103006@stu.sicau.edu.cn;
2020103005@stu.sicau.edu.cn; 13577@sicau.edu.cn; 41187@sicau.edu.cn;
13643@sicau.edu.cn; 10334@sicau.edu.cn 10.3390/nano12091534 2022 12 9
- - 1534 -
Gao, Yahui; Ma, Li; Liu, George Initial Analysis of Structural Variation
Detections in Cattle Using Long-Read Sequencing Methods Genes EN Article
 cattle; structural variation; long-read sequencing Structural variations
(SVs), as a great source of genetic variation, are widely distributed in the
genome. SVs involve longer genomic sequences and potentially have stronger effects
than SNPs, but they are not well captured by short-read sequencing owing to their
size and relevance to repeats. Improved characterization of SVs can provide more
advanced insight into complex traits. With the availability of long-read
sequencing, it has become feasible to uncover the full range of SVs. Here, we
sequenced one cattle individual using 10× Genomics (10 × G) linked
read, Pacific Biosciences (PacBio) continuous long reads (CLR) and circular
consensus sequencing (CCS), as well as Oxford Nanopore Technologies (ONT)
PromethION. We evaluated the ability of various methods for SV detection. We
identified 21,164 SVs, which amount to 186 Mb covering 7.07% of the whole genome.
The number of SVs inferred from long-read-based inferences was greater than that
from short reads. The PacBio CLR identified the most of large SVs and covered the
most genomes. SVs called with PacBio CCS and ONT data showed high uniformity. The
one with the most overlap with the results obtained by short-read data was PB CCS.
Together, we found that long reads outperformed short reads in terms of SV
detections. Animal Genomics and Improvement Laboratory, Beltsville Agricultural
Research Center, Agricultural Research Service, U.S. Department of Agriculture,
Beltsville, MD 20705, USA gyhalvin@gmail.com; lima@umd.edu; george.liu@usda.gov
10.3390/genes13050828 2022 13 5 - - 828 -
Avetyan, Diana; Hakobyan, Siras; Nikoghosyan, Maria; Ghukasyan, Lilit; Khachatryan,
Gisane; Sirunyan, Tamara; Muradyan, Nelli; Zakharyan, Roksana; Chavushyan,
Andranik; Hayrapetyan, Varduhi; Hovhannisyan, Anahit; Mohamed Bakhash, Shah;
Jerome, Keith; Roychoudhury, Pavitra; Greninger, Alexander; Niazyan, Lyudmila;
Davidyants, Mher; Melik-Andreasyan, Gayane; Sargsyan, Shushan; Nersisyan, Lilit;
Arakelyan, Arsen Molecular Analysis of SARS-CoV-2 Lineages in Armenia Viruses
EN Article COVID-19; SARS-CoV-2; coronavirus; nanopore sequencing;
Illumina sequencing; whole-genome sequencing; Armenia The sequencing of SARS-CoV-2
provides essential information on viral evolution, transmission, and epidemiology.
In this paper, we performed the whole-genome sequencing of SARS-CoV-2 using
nanopore and Illumina sequencing to describe the circulation of the virus lineages
in Armenia. The analysis of 145 full genomes identified six clades (19A, 20A, 20B,
20I, 21J, and 21K) and considerable intra-clade PANGO lineage diversity.
Phylodynamic and transmission analysis allowed to attribute specific clades as well
as infer their importation routes. Thus, the first two waves of positive case
increase were caused by the 20B clade, the third peak caused by the 20I (Alpha),
while the last two peaks were caused by the 21J (Delta) and 21K (Omicron) variants.
The functional analyses of mutations in sequences largely affected epitopes
associated with protective HLA loci and did not cause the loss of the signal in PCR
tests targeting ORF1ab and N genes as confirmed by RT-PCR. We also compared the
performance of nanopore and Illumina short-read sequencing and showed the utility
of nanopore sequencing as an efficient and affordable alternative for large-scale
molecular epidemiology research. Thus, our paper describes new data on the genomic
diversity of SARS-CoV-2 variants in Armenia in the global context of the virus
molecular genomic surveillance. Laboratory of Human Genomics, Institute of
Molecular Biology NAS RA, Yerevan 0014, Armenia d_avetyan@mb.sci.am;
s_hakobyan@mb.sci.am; m_nikoghosyan@mb.sci.am; l_ghukasyan@mb.sci.am;
g_khachatryan@mb.sci.am; t_sirunyan@mb.sci.am; n_muradyan@mb.sci.am;
roksana.zakharyan@rau.am; a_chavushyan@mb.sci.am; haivard@mail.ru;
hovhannisyananahit19@gmail.com; shahm4@uw.edu; kjerome@fredhutch.org;
proychou@fredhutch.org; agrening@uw.edu; lyudmila.niazyan@gmail.com;
davidyants@gmail.com; gayane.melik-andreasyan@ncdc.am; premier_h@yahoo.com;
lilit.nersisyan@ki.se; aarakelyan@sci.am 10.3390/v14051074 2022 14 5 -
- 1074 -
Amoutzias, Grigorios; Nikolaidis, Marios; Hesketh, Andrew The Notable Achievements
and the Prospects of Bacterial Pathogen Genomics Microorganisms EN
Perspective bacterial pathogen; genomics; Illumina; Pacific Biosciences;
Oxford Nanopore; evolution; forensics; food-borne pathogens; clinical microbiology;
metagenome-assembled genomes Throughout the entirety of human history, bacterial
pathogens have played an important role and even shaped the fate of civilizations.
The application of genomics within the last 27 years has radically changed the way
we understand the biology and evolution of these pathogens. In this review, we
discuss how the short- (Illumina) and long-read (PacBio, Oxford Nanopore)
sequencing technologies have shaped the discipline of bacterial pathogen genomics,
in terms of fundamental research (i.e., evolution of pathogenicity), forensics,
food safety, and routine clinical microbiology. We have mined and discuss some of
the most prominent data/bioinformatics resources such as NCBI pathogens, PATRIC,
and Pathogenwatch. Based on this mining, we present some of the most popular
sequencing technologies, hybrid approaches, assemblers, and annotation pipelines. A
small number of bacterial pathogens are of very high importance, and we also
present the wealth of the genomic data for these species (i.e., which ones they
are, the number of antimicrobial resistance genes per genome, the number of
virulence factors). Finally, we discuss how this discipline will probably be
transformed in the near future, especially by transitioning into metagenome-
assembled genomes (MAGs), thanks to long-read sequencing. Bioinformatics
Laboratory, Department of Biochemistry and Biotechnology, University of Thessaly,
41500 Larissa, Greece amoutzias@bio.uth.gr; marionik23@gmail.com;
a.hesketh@brighton.ac.uk 10.3390/microorganisms10051040 2022 10 5
- - 1040 -
Hai, Dao; Yen, Duong; Liem, Pham; Tam, Bui; Huong, Do; Hang, Bui; Hieu, Dang;
Garigliany, Mutien-Marie; Coppieters, Wouter; Kestemont, Patrick; Phuong, Nguyen;
Farnir, Frédéric A High-Quality Genome Assembly of Striped Catfish (Pangasianodon
hypophthalmus) Based on Highly Accurate Long-Read HiFi Sequencing Data Genes EN
Article striped catfish; chromosome-scale genome assembly; selective
breeding; HiFi reads The HiFi sequencing technology yields highly accurate long-
read data with accuracies greater than 99.9% that can be used to improve results
for complex applications such as genome assembly. Our study presents a high-quality
chromosome-scale genome assembly of striped catfish (Pangasianodon hypophthalmus),
a commercially important species cultured mainly in Vietnam, integrating HiFi reads
and Hi-C data. A 788.4 Mb genome containing 381 scaffolds with an N50 length of
21.8 Mb has been obtained from HiFi reads. These scaffolds have been further
ordered and clustered into 30 chromosome groups, ranging from 1.4 to 57.6 Mb, based
on Hi-C data. The present updated assembly has a contig N50 of 14.7 Mb,
representing a 245-fold and 4.2-fold improvement over the previous Illumina and
Illumina-Nanopore-Hi-C based version, respectively. In addition, the proportion of
repeat elements and BUSCO genes identified in our genome is remarkably higher than
in the two previously released striped catfish genomes. These results highlight the
power of using HiFi reads to assemble the highly repetitive regions and to improve
the quality of genome assembly. The updated, high-quality genome assembled in this
work will provide a valuable genomic resource for future population genetics,
conservation biology and selective breeding studies of striped catfish.
FARAH/Sustainable Animal Production, Faculty of Veterinary Medicine,
University of Liege (B43), 4000 Liege, Belgium dmhai@ctu.edu.vn;
thuyyen@ctu.edu.vn; ptliem@ctu.edu.vn; bmtam@ctu.edu.vn; dtthuong@ctu.edu.vn;
btbhang@ctu.edu.vn; quanghieudang.87@gmail.com; mmgarigliany@uliege.be;
wouter.coppieters@uliege.be; patrick.kestemont@unamur.be; ntphuong@ctu.edu.vn;
f.farnir@uliege.be 10.3390/genes13050923 2022 13 5 - - 923
-
Gomes-de-Sá, Sónia; Barradas, Patrícia; Queirós-Reis, Luís; Matas, Isabel; Amorim,
Irina; Cardoso, Luís; Muñoz-Mérida, Antonio; Mesquita, JoãoDe Novo Assembly of the
Dirofilaria immitis Genome by Long-Read Nanopore-Based Sequencing Technology on an
Adult Worm from a Canine Cardiopulmonary Dirofilariosis Case Animals EN
Communication Dirofilaria immitis; genome; macrocyclic lactone
resistance; long-read Dirofilaria immitis is a zoonotic parasitic nematode that
infects domestic and wild canids, among its vertebrate hosts. The genetic analysis
of D. immitis nowadays transcends the need for genetic taxonomy of nematodes, such
as the study of resistance to macrocyclic lactone. We expanded the use of long-read
nanopore-based sequencing technology on nematodes by performing genomic de novo
assembly of a D. immitis specimen retrieved from a canine cardiopulmonary
dirofilariasis case using the ONT MinION platform, followed by the study of
macrocyclic lactone resistance. The assembled genome of D. immitis consists of 110
contigs with an N50 of 3687191. The genome size is 87899012 and contains a total of
9741 proteins; 6 ribosomal RNAs, with three belonging to the small subunit (18S)
and three to the large subunit (28S); and 73 tRNAs. Subsequent analysis of six loci
previously characterized as being associated to macrocyclic lactone resistance
selection pressure showed that four have a genotype associated with either some
loss of efficacy or the resistance phenotype. Considering the zoonotic potential of
D. immitis, the identification of a resistant parasite alerts for the overuse of
macrocyclic lactone in the region, which poses a potential risk to both veterinary
and human public health. ICBAS—School of Medicine and Biomedical Sciences,
Porto University, 4050-313 Porto, Portugal soniavilargomessa@gmail.com;
patricia.barradas@iucs.cespu.pt; luisqueirosreis@gmail.com; isa_mmcc@hotmail.com;
ifamorim@icbas.up.pt; lcardoso@utad.pt; amunoz@cibio.up.pt; jrmesquita@icbas.up.pt
10.3390/ani12111342 2022 12 11 - - 1342 -
Fan, Xin; Zhang, Beibei; Fan, Lijun; Chen, Jiajia; Su, Chang; Cao, Bingyan; Wei,
Liya; Qin, Miao; Gong, Chunxiu DNA Hypermethylation and a Specific Methylation
Spectrum on the X Chromosome in Turner Syndrome as Determined by Nanopore
Sequencing Journal of Personalized Medicine EN Article Turner syndrome;
methylation; nanopore sequencing The molecular genetic mechanism of Turner
syndrome (TS) still leaves much to be discovered. Methods: TS (45X0) patients and
age-matched controls (46XX and 46XY) were selected. The nanopore sequencing
combined with trio-whole exome sequencing (trio-WES) were used for the first time
to investigate TS. Results: Thirteen TS (45X0) patients and eight controls were
enrolled. Trio-WES analysis did not find any pathogenetic or likely pathogenic
variants except X chromosome (chrX) deletion. The average methylation levels and
patterns of chrX in 45X0 and 46XY were similar, and significantly higher than in
46XX (p = 2.22 × 10−16). Both hyper-methylation and hypo-methylation
were detected in the CpG island (CGI), CGI_shore, promoter, genebody, and PAR1-
region, while in the transposon element inactivation regions of the chrX and
hypermethylation were predominant. A total of 125 differentially methylated genes
were identified in 45X0 compared to 46XX, including 8 and 117 hypermethylated and
hypomethylated genes, respectively, with the enrichment terms of mitophagy,
regulation of DNA-binding transcription factor activity, etc. Conclusions: The
results suggest that the methylation profile in patients with TS might be
determined by the number of X chromosomes; the patterns of methylation in TS were
precisely associated with the maintenance of genomic stability and improvement of
gene expression. Differentially methylated genes/pathways might reveal the
potential epigenetic modulation and lead to better understanding of TS.
Department of Endocrinology, Genetics and Metabolism, National Center for
Children’s Health, Beijing Children’s Hospital, Capital Medical University, Beijing
100045, China fanxin602@163.com; aazhangbei@126.com; fanlijun0916@163.com;
chenjiaj2009@126.com; changsulucky@yahoo.com; caoby1982@163.com; wly0830@sina.com;
oqinmiaoo@126.com; chunxiugong@sina.com 10.3390/jpm12060872 2022 12 6
- - 872 -
Rasmussen, Astrid; Hildonen, Mathis; Vissing, John; Duno, Morten; Tümer, Zeynep;
Birkedal, Ulf High Resolution Analysis of DMPK Hypermethylation and Repeat
Interruptions in Myotonic Dystrophy Type 1 Genes EN Article Oxford
nanopore; long-read sequencing; DM1; epigenetics; methylation; diagnostics; Cas9
Myotonic dystrophy type 1 (DM1) is a multisystemic neuromuscular disorder
caused by the expansion of a CTG repeat in the 3′-UTR of DMPK, which is
transcribed to a toxic gain-of-function RNA that affects splicing of a range of
genes. The expanded repeat is unstable in both germline and somatic cells. The
variable age at disease onset and severity of symptoms have been linked to the
inherited CTG repeat length, non-CTG interruptions, and methylation levels flanking
the repeat. In general, the genetic biomarkers are investigated separately with
specific methods, making it tedious to obtain an overall characterisation of the
repeat for a given individual. In the present study, we employed Oxford nanopore
sequencing in a pilot study to simultaneously determine the repeat lengths,
investigate the presence and nature of repeat interruptions, and quantify
methylation levels in the regions flanking the CTG-repeats in four patients with
DM1. We determined the repeat lengths, and in three patients, we observed
interruptions which were not detected using repeat-primed PCR. Interruptions may
thus be more common than previously anticipated and should be investigated in
larger cohorts. Allele-specific analyses enabled characterisation of aberrant
methylation levels specific to the expanded allele, which greatly increased the
sensitivity and resolved cases where the methylation levels were ambiguous.
Kennedy Center, Department of Clinical Genetics, Copenhagen University
Hospital, Rigshospitalet, 2600 Glostrup, Denmark rasmussenastrid4@gmail.com;
mathis.hildonen@regionh.dk; john.vissing@regionh.dk; morten.dunoe@regionh.dk;
zeynep.tumer@regionh.dk; ulf.birkedal@regionh.dk 10.3390/genes13060970 2022
13 6 - - 970 -
Padane, Abdou; Diedhiou, Cyrille; Gueye, Khadim; Ndiour, Samba; Diagne, Ndéye;
Mboup, Aminata; Mbow, Moustapha; Lo, Cheikh; Leye, Nafissatou; Ndoye, Aissatou;
Ndiaye, Anna; Ndiaye, Seyni; Dia, Yacine; Lo, Gora; Wade, Djibril; Ahouidi,
Ambroise; Diaw, Papa; Sarr, Marièma; Beye, Mamadou; Kaba, Lanceï; Cissé, Badara;
Sokhna, Cheikh; Camara, Makhtar; Kane, Ndéye; Mboup, Souleymane Dynamics of
Variants of Concern (VOC) of SARS-CoV-2 during the Different Waves of COVID-19 in
Senegal COVID EN Article COVID-19; SARS-CoV-2; VOC; genome;
phylodynamics; Senegal Background: In Senegal, the incidence of SARS-CoV-2 evolved
with four successive epidemic waves. The first wave started in March 2020 with low
virus variability, whilst the second outbreak, which started in December 2020, was
dominated by the Alpha variant. The third wave took place in June 2021, and the
fourth at the end of November 2021. Our interest was to investigate the involvement
of variants of concern during these four waves and to track the viral diversity of
SARS-CoV-2. Methodology: During the four waves of the pandemic, 276,876
nasopharyngeal swabs were analyzed at the Institut de Recherche en Santé, de
Surveillance Epidémiologique et de Formation (IRESSEF). Of these, 22,558
samples tested positive for SARS-CoV-2 by RT-PCR. Then, the virus genomes were
sequenced in 817 positive samples using the ARTIC Network of Oxford Nanopore
Technologies (ONT). In addition, 10% of the negative samples in RT-PCR new variants
were also targeted for the detection of new and previously undescribed variants.
Results: Our data have overall shown that the Senegalese strains are very similar
to each other or closely related to other strains, such as Gambia, France etc.
During the first wave, the most common clade found was 19A (67.5%) and a majority
of the samples were of the B.1 (50%) lineage. We noted more diversity during the
second wave where clade 20A (38.4%) was more frequent, followed by clade 20B
(31.52%) and 20I (9.74%). At the level of lineages, we identified variants of
concern as B.1.1.7 (9.74%) and B.1.617.2 (0.86%). In the third wave, we observed at
the clade level with mainly 21A (32.63%) and 21J (16.84%). During the fourth wave
at the end of November 2021, we mainly identified clade 21K Omicron variant 21K
(B.1.1.529 and BA.1) (80.47%) and Delta variant (21A, 21J, and 21I) (AY.103,
AY.122, AY.122.1, AY.26, AY.34, AY.36, AY.4, AY.48, AY.57, AY.61, and AY.87)
(14.06%). Impact: SARS-CoV-2 diversity may affect the virus’s properties,
such as how it spreads, disease severity, or the performance of vaccines, tools, or
other public health and social measures. Therefore, such tracking of SARS-CoV-2
variants is not only of public interest, but also highlights the role some African
institutes such as IRESSEF with surveillance capabilities through the real-time
sequencing of SARS-CoV-2 genomes in the local context. Conclusion: In Senegal, the
SARS-CoV-2 pandemic has disrupted the organization of the health system. IRESSEF
contributed to put in place strategies to respond effectively to the expectations
of medical authorities by providing them with data on the strains circulating in
Senegal at each moment of the epidemic. Institut de Recherche en Santé, de
Surveillance Épidémiologique et de Formation (IRESSEF), Dakar BP 7325, Senegal
abdou.padane@iressef.org; cyrille.diedhiou@iressef.org;
khadim.gueye@iressef.org; samba.ndiour@iressef.org; diabou.diagne@iressef.org;
aminata.mboup@iressef.org; moustapha.mbow@iressef.org; cibrahimalo@gmail.com;
nafissatou.leye@iressef.org; aissatou.sow@iressef.org;
annajulienne.ndiaye@iressef.org; seynindiaye07@gmail.com; yassine.dia@iressef.org;
gora.lo@iressef.org; djibril.wade@iressef.org; ambroise.ahouidi@iressef.org;
papaalassane.diaw@iressef.org; sarrmarem@yahoo.fr; bemamadou@gmail.com;
lancekaba1@gmail.com; badara.cisse@iressef.org; cheikh.sokhna@ird.fr;
makhtar.camara@iressef.org; coumba.toure@iressef.org; souleymane.mboup@iressef.org
10.3390/covid2060052 2022 2 6 - - 52 -
Azagi, Tal; Dirks, Ron; Yebra-Pimentel, Elena; Schaap, Peter; Koehorst, Jasper;
Esser, Helen; Sprong, Hein Assembly and Comparison of Ca. Neoehrlichia
mikurensis Genomes Microorganisms EN Article tick-borne pathogens;
Ixodes ricinus; Nanopore sequencing; rickettsiales; Anaplasmataceae Ca.
Neoehrlichia mikurensis is widely prevalent in I. ricinus across Europe and has
been associated with human disease. However, diagnostic modalities are limited, and
much is still unknown about its biology. Here, we present the first complete Ca.
Neoehrlichia mikurensis genomes directly derived from wildlife reservoir host
tissues, using both long- and short-read sequencing technologies. This pragmatic
approach provides an alternative to obtaining sufficient material from clinical
cases, a difficult task for emerging infectious diseases, and to expensive and
challenging bacterial isolation and culture methods. Both genomes exhibit a larger
chromosome than the currently available Ca. Neoehrlichia mikurensis genomes and
expand the ability to find new targets for the development of supportive laboratory
diagnostics in the future. Moreover, this method could be utilized for other tick-
borne pathogens that are difficult to culture. Centre for Infectious Diseases
Research, National Institute for Public Health and the Environment, 3720 BA
Bilthoven, The Netherlands tal.azagi@rivm.nl; dirks@futuregenomics.tech; yebra-
pimentel@futuregenomics.tech; peter.schaap@wur.nl; jasper.koehorst@wur.nl;
helen.esser@wur.nl; hein.sprong@rivm.nl 10.3390/microorganisms10061134 2022
10 6 - - 1134 -
Mastriani, Emilio; Bienes, Kathrina; Wong, Gary; Berthet, Nicolas PIMGAVir and
Vir-MinION: Two Viral Metagenomic Pipelines for Complete Baseline Analysis of 2nd
and 3rd Generation Data Viruses EN Technical Note taxonomic
classification; metagenomic pipeline; 2nd and 3rd sequencing generation; multiple
strategies analysis The taxonomic classification of viral sequences is
frequently used for the rapid identification of pathogens, which is a key point for
when a viral outbreak occurs. Both Oxford Nanopore Technologies (ONT) MinION and
the Illumina (NGS) technology provide efficient methods to detect viral pathogens.
Despite the availability of many strategies and software, matching them can be a
very tedious and time-consuming task. As a result, we developed PIMGAVir and Vir-
MinION, two metagenomics pipelines that automatically provide the user with a
complete baseline analysis. The PIMGAVir and Vir-MinION pipelines work on 2nd and
3rd generation data, respectively, and provide the user with a taxonomic
classification of the reads through three strategies: assembly-based, read-based,
and clustering-based. The pipelines supply the scientist with comprehensive results
in graphical and textual format for future analyses. Finally, the pipelines equip
the user with a stand-alone platform with dedicated and various viral databases,
which is a requirement for working in field conditions without internet connection.
Unit of Discovery and Molecular Characterization of Pathogens, Centre for
Microbes, Development and Health, Institut Pasteur of Shanghai, Chinese Academy of
Sciences, Shanghai 200031, China emiliomastrani@icloud.com; kathrina@ips.ac.cn;
garyckwong@ips.ac.cn; nicolas.berthet@pasteur.fr 10.3390/v14061260 2022 14
6 - - 1260 -
Lei, Xin; Zhang, Jiayan; Hong, Hao; Yuan, Zhishan; Liu, Zewen Controllable
Shrinking Fabrication of Solid-State Nanopores Micromachines EN Review
solid-state nanopores; shrinking fabrication; size and shape control; high
energy beam Nanopores have attracted widespread attention in DNA sequencing and
protein or biomarker detection, owning to the single-molecule-scale detection
accuracy. Despite the most use of naturally biological nanopores before, solid-
state nanopores are widely developed with strong robustness, controllable sizes and
geometries, a wide range of materials available, as well as flexible manufacturing.
Therefore, various techniques typically based on focused ion beam or electron beam
have been explored to drill nanopores directly on free-standing nanofilms. To
further reduce and sculpt the pore size and shape for nano or sub-nano space-time
sensing precision, various controllable shrinking technologies have been employed.
Correspondingly, high-energy-beam-induced contraction with direct visual feedback
represents the most widely used. The ability to change the pore diameter was
attributed to surface tension induced original material migration into the nanopore
center or new material deposition on the nanopore surface. This paper reviews
typical solid-state nanopore shrinkage technologies, based on the careful summary
of their principles and characteristics in particularly size and morphology
changes. Furthermore, the advantages and disadvantages of different methods have
also been compared completely. Finally, this review concludes with an optimistic
outlook on the future of solid-state nanopores. School of Chemistry, Beihang
University, Beijing 100191, China leixin@buaa.edu.cn; zhangjiayan@buaa.edu.cn;
honghao@tsinghua.edu.cn; zhishanyuan@gdut.edu.cn; liuzw@tsinghua.edu.cn
10.3390/mi13060923 2022 13 6 - - 923 -
Pogka, Vasiliki; Papadopoulou, Gethsimani; Valiakou, Vaia; Sgouras, Dionyssios;
Mentis, Andreas; Karamitros, Timokratis Targeted Virome Sequencing Enhances
Unbiased Detection and Genome Assembly of Known and Emerging Viruses—The
Example of SARS-CoV-2 Viruses EN Article target enrichment; virome
sequencing; SARS-CoV-2; COVID-19; NGS diagnostics; emerging viruses; nanopore
sequencing Targeted virome enrichment and sequencing (VirCapSeq-VERT) utilizes a
pool of oligos (baits) to enrich all known—up to 2015—vertebrate-
infecting viruses, increasing their detection sensitivity. The hybridisation of the
baits to the target sequences can be partial, thus enabling the detection and
genomic reconstruction of novel pathogens with <40% genetic diversity compared
to the strains used for the baits’ design. In this study, we deploy this
method in multiplexed mixes of viral extracts, and we assess its performance in the
unbiased detection of DNA and RNA viruses after cDNA synthesis. We further assess
its efficiency in depleting various background genomic material. Finally, as a
proof-of-concept, we explore the potential usage of the method for the
characterization of unknown, emerging human viruses, such as SARS-CoV-2, which may
not be included in the baits’ panel. We mixed positive samples of equimolar
DNA/RNA viral extracts from SARS-CoV-2, coronavirus OC43, cytomegalovirus,
influenza A virus H3N2, parvovirus B19, respiratory syncytial virus, adenovirus C
and coxsackievirus A16. Targeted virome enrichment was performed on a dsDNA mix,
followed by sequencing on the NextSeq500 (Illumina) and the portable MinION
sequencer, to evaluate its usability as a point-of-care (PoC) application. Genome
mapping assembly was performed using viral reference sequences. The untargeted
libraries contained less than 1% of total reads mapped on most viral genomes, while
RNA viruses remained undetected. In the targeted libraries, the percentage of
viral-mapped reads were substantially increased, allowing full genome assembly in
most cases. Targeted virome sequencing can enrich a broad range of viruses,
potentially enabling the discovery of emerging viruses. Laboratory of Medical
Microbiology, Department of Microbiology, Hellenic Pasteur Institute, 11521 Athens,
Greece vpoga@pasteur.gr; gesthpap@pasteur.gr; vanessa.valiakou@gmail.com;
sgouras@pasteur.gr; mentis@pasteur.gr; tkaram@pasteur.gr 10.3390/v14061272 2022
14 6 - - 1272 -
Tombácz, Dóra; Kakuk, Balázs; Torma, Gábor; Csabai, Zsolt; Gulyás, Gábor; Tamás,
Vivien; Zádori, Zoltán; Jefferson, Victoria; Meyer, Florencia; Boldogkői, Zsolt
In-Depth Temporal Transcriptome Profiling of an Alphaherpesvirus Using
Nanopore Sequencing Viruses EN Article herpesviruses; bovine
alphaherpesvirus type 1; transcriptome; transcript isoforms; long-read sequencing;
nanopore sequencing; direct cDNA sequencing; transcription start site;
transcription end site In this work, a long-read sequencing (LRS) technique based
on the Oxford Nanopore Technology MinION platform was used for quantifying and
kinetic characterization of the poly(A) fraction of bovine alphaherpesvirus type 1
(BoHV-1) lytic transcriptome across a 12-h infection period. Amplification-based
LRS techniques frequently generate artefactual transcription reads and are biased
towards the production of shorter amplicons. To avoid these undesired effects, we
applied direct cDNA sequencing, an amplification-free technique. Here, we show that
a single promoter can produce multiple transcription start sites whose distribution
patterns differ among the viral genes but are similar in the same gene at different
timepoints. Our investigations revealed that the circ gene is expressed with
immediate–early (IE) kinetics by utilizing a special mechanism based on the
use of the promoter of another IE gene (bicp4) for the transcriptional control.
Furthermore, we detected an overlap between the initiation of DNA replication and
the transcription from the bicp22 gene, which suggests an interaction between the
two molecular machineries. This study developed a generally applicable LRS-based
method for the time-course characterization of transcriptomes of any organism.
Department of Medical Biology, Albert Szent-Györgyi Medical School,
University of Szeged, Somogyi u. 4, 6720 Szeged, Hungary tombacz.dora@med.u-
szeged.hu; kakuk.balazs@med.u-szeged.hu; torma.gabor@med.u-szeged.hu;
csabai.zsolt@med.u-szeged.hu; gulyas.gabor@med.u-szeged.hu;
tamas.vivien@agrar.mta.hu; zadori.zoltan@agrar.mta.hu; vaj13@msstate.edu;
florencia.meyer@msstate.edu; boldogkoi.zsolt@med.u-szeged.hu 10.3390/v14061289
2022 14 6 - - 1289 -
Cumbo, Cosimo; Minervini, Crescenzio; Albano, Francesco Third-Generation
Sequencing in Clinical Practice: The New Era of Precision Medicine? Applied
Sciences EN Commentary third-generation sequencing; nanopore sequencing;
clinical practice; infectious diseases; inherited disorders; cancer diseases In the
last decades, the spreading of next-generation sequencing (NGS) in clinical
practice has considerably increased the genomic knowledge of several disorders. The
recent advent of third-generation sequencing is transforming the standard way of
conceiving clinical genomics, overcom-ing the main limits of conventional NGS
technologies and achieving challenges so far considered unreasonable. What was
impracticable only a few years ago, in terms of potential and affordability, now is
becoming achievable. The new sequencing era will improve diagnostic and therapeutic
ap-proaches, providing clinicians with valid support in their practice.
Hematology and Stem Cell Transplantation Unit, Department of Emergency and
Organ Transplantation (D.E.T.O.), University of Bari “Aldo Moro”, 70124 Bari, Italy
cosimo.cumbo@uniba.it; eziominervini@gmail.com; francesco.albano@uniba.it
10.3390/app12126058 2022 12 12 - - 6058 -
Lanszki, Zsófia; Lanszki, József; Tóth, Gábor; Zeghbib, Safia; Jakab, Ferenc;
Kemenesi, Gábor Retrospective Detection and Complete Genomic Sequencing of Canine
morbillivirus in Eurasian Otter (Lutra lutra) Using Nanopore Technology
Viruses EN Communication Mustelidae; NGS; third generation
sequencing; conservation biology; MinION; enzootic The Eurasian otter (Lutra
lutra) is a piscivorous apex predator in aquatic habitats, and a flagship species
of conservation biology throughout Europe. Despite the wide distribution and
ecological relevance of the species, there is a considerable lack of knowledge
regarding its virological and veterinary health context, especially in Central
Europe. Canine morbillivirus (Canine distemper virus (CDV)) is a highly contagious
viral agent of the family Paramyxoviridae with high epizootic potential and
veterinary health impact. CDV is present worldwide among a wide range of animals;
wild carnivores are at particular risk. As part of a retrospective study, lung-
tissue samples (n = 339) from Eurasian otters were collected between 2000 and 2021
throughout Hungary. The samples were screened for CDV using a real-time RT-PCR
method. Two specimens proved positive for CDV RNA. In one sample, the complete
viral genome was sequenced using a novel, pan-genotype CDV-specific amplicon-based
sequencing method with Oxford Nanopore sequencing technology. Both viral sequences
were grouped to a European lineage based on the hemagglutinin-gene phylogenetic
classification. In this article, we present the feasibility of road-killed animal
samples for understanding the long-term dynamics of CDV among wildlife and provide
novel virological sequence data to better understand CDV circulation and evolution.
National Laboratory of Virology, Szentágothai Research Centre, University of
Pécs, 7624 Pécs, Hungary lanszkizsofi@gmail.com; lanszkij@gmail.com;
toth.gabor.endre@gmail.com; zeghbib.safia@gmail.com; jakab.ferenc@pte.hu;
kemenesi.gabor@gmail.com 10.3390/v14071433 2022 14 7 - - 1433
-
Ndotono, Evalyne; Khamis, Fathiya; Bargul, Joel; Tanga, Chrysantus Insights
into the Gut Microbial Communities of Broiler Chicken Fed Black Soldier Fly Larvae-
Desmodium-Based Meal as a Dietary Protein Source Microorganisms EN
Article black soldier fly; gut microbiota; poultry feed; broiler chicken;
Oxford nanopore sequencing The utilization of insect-based diets to improve
gastrointestinal function and gut health in poultry is gaining global attention as
a promising feed additive. The objective of this study was to determine the optimal
inclusion level of the full-fat black soldier fly larvae (BSFL) and Desmodium
intortum (DI) in broiler chicken diets and to evaluate their impact on the
microbial community in the gut. The bacterial communities were characterized using
Oxford nanopore sequencing of the full-length bacterial 16S rRNA gene. Four dietary
treatments, T1 (25% DI + 75% BSFL), T2 (50% DI + 50% BSFL), T3 (75% DI + 25% BSFL)
and T4 (100% fishmeal + 0% DI + BSFL), were fed to the broiler chickens for a
period of 42 days. Out of the 395,034 classified reads analyzed, the most
predominant phyla identified across all the four dietary treatments were Firmicutes
(94%), Bacteroidetes (3%), and Proteobacteria (2%). The T1 diet showed the highest
alpha diversity and richness according to the Chao1 and Shannon indices. Beta
diversity assessment revealed a significant influence of diet on the abundance of
the microbiome. There was an increase in beneficial lactic acid bacteria with
increasing inclusion of BSFL in the diets. Our findings strongly support the
inclusion of BSFL into poultry diet as a promising protein source to reshape the
gut microbiota for improved gut health, immune response, and food safety.
International Centre of Insect Physiology and Ecology (ICIPE), Nairobi P.O.
Box 30772-00100, Kenya endotono@icipe.org; fkhamis@icipe.org; jbargul@icipe.org;
ctanga@icipe.org 10.3390/microorganisms10071351 2022 10 7 - -
1351 -
Rabbachin, Laura; Piñar, Guadalupe; Nir, Irit; Kushmaro, Ariel; Pavan, Mariela;
Eitenberger, Elisabeth; Waldherr, Monika; Graf, Alexandra; Sterflinger, Katja
A Multi-Analytical Approach to Infer Mineral–Microbial Interactions
Applied to Petroglyph Sites in the Negev Desert of Israel Applied Sciences EN
Article petroglyphs; Negev desert; biodeterioration; nanopore sequencing
technology; metagenomics; analytical techniques Petroglyph sites exist all over the
world. They are one of the earliest forms of mankind’s expression and a
precursor to art. Despite their outstanding value, comprehensive research on
conservation and preservation of rock art is minimal, especially as related to
biodeterioration. For this reason, the main objective of this study was to explore
the factors involved in the degradation of petroglyph sites in the Negev desert of
Israel, with a focus on biodegradation processes. Through the use of culture-
independent microbiological methods (metagenomics), we characterized the
microbiomes of the samples, finding they were dominated by bacterial communities,
in particular taxa of Actinobacteria and Cyanobacteria, with resistance to
radiation and desiccation. By means of XRF and Raman spectroscopies, we defined the
composition of the stone (calcite and quartz) and the dark crust (clay minerals
with Mn and Fe oxides), unveiling the presence of carotenoids, indicative of
biological colonization. Optical microscopy and SEM–EDX analyses on thin
sections highlighted patterns of weathering, possibly connected to the presence of
biodeteriorative microorganisms that leach the calcareous matrix from the bedrock
and mobilize metal cations from the black varnish for metabolic processes, slowly
weathering it. Institute of Natural Sciences and Technology in the Arts (INTK),
Academy of Fine Arts Vienna, Schillerplatz 3, 1010 Vienna, Austria
l.rabbachin@akbild.ac.at; g.pinarlarrubia@akbild.ac.at; irin@post.bgu.ac.il;
arielkus@bgu.ac.il; marielap@bgu.ac.il; elisabeth.eitenberger@tuwien.ac.at;
monika.waldherr@fh-campuswien.ac.at; alexandra.graf@fh-campuswien.ac.at;
k.sterflinger@akbild.ac.at 10.3390/app12146936 2022 12 14 - -
6936 -
Ryan, Yan; Harrison, Abbie; Trivett, Hannah; Hartley, Catherine; David, Jonathan;
Clark, Graeme; Hiscox, Julian RIPpore: A Novel Host-Derived Method for the
Identification of Ricin Intoxication through Oxford Nanopore Direct RNA Sequencing
Toxins EN Article ricin; RNA sequencing; MinIon; ribosome
inactivating proteins; saporin; ribosomes Ricin is a toxin which enters cells and
depurinates an adenine base in the sarcin-ricin loop in the large ribosomal
subunit, leading to the inhibition of protein translation and cell death. We
postulated that this depurination event could be detected using Oxford Nanopore
Technologies (ONT) direct RNA sequencing, detecting a change in charge in the ricin
loop. In this study, A549 cells were exposed to ricin for 2–24 h in order to
induce depurination. In addition, a novel software tool was developed termed
RIPpore that could quantify the adenine modification of ribosomal RNA induced by
ricin upon respiratory epithelial cells. We provided demonstrable evidence for the
first time that this base change detected is specific to RIP activity using a
neutralising antibody against ricin. We believe this represents the first detection
of depurination in RNA achieved using ONT sequencers. Collectively, this work
highlights the potential for ONT and direct RNA sequencing to detect and quantify
depurination events caused by ribosome-inactivating proteins such as ricin. RIPpore
could have utility in the evaluation of new treatments and/or in the diagnosis of
exposure to ricin. Institute for Infection, Veterinary and Ecological
Sciences, University of Liverpool, Liverpool L3 5RF, UK yryan@liv.ac.uk;
psaharri@liverpool.ac.uk; h.trivett@liverpool.ac.uk;
catherine.hartley@liverpool.ac.uk; jdavid@mail.dstl.gov.uk; gcclark@dstl.gov.uk;
julian.hiscox@liverpool.ac.uk 10.3390/toxins14070470 2022 14 7 - -
470 -
Alai, Shweta; Gautam, Manish; Palkar, Sonali; Oswal, Jitendra; Gairola, Sunil;
Dhotre, Dhiraj Characterization of Bordetella pertussis Strains Isolated from
India Pathogens EN Article genetic divergence; whooping cough; developing
country; allele; genotype; phylogeny; Oxford sequencing; vaccines Despite high
level vaccination and the availability of two different types of vaccines, whole
cell (wP) and acellular vaccines (aP), the resurgence of pertussis has been
reported in many countries. Antigenic variation within circulating and vaccine
strains is the most documented reason reported for the resurgence of pertussis.
Research on genetic divergence among circulating and vaccine strains has largely
been reported in countries using aP vaccines. There are inadequate data available
for antigenic variation in B. pertussis from wP-using countries. India has used wP
for more than 40 years in their primary immunization program. The present study
reports five clinical isolates of B. pertussis from samples of pediatric patients
with pertussis symptoms observed in India. Genotypic and phenotypic
characterization of clinical isolates were performed by serotyping, genotyping,
whole genome analyses and comparative genomics. All clinical isolates showed
serotype 1, 2 and 3 based on the presence of fimbriae 2 and 3. Genotyping showed
genetic similarities in allele types for five aP genes within vaccine strains and
clinical isolates reported from India. The presence of the ptxP3 genotype was
observed in two out of five clinical isolates. Whole-genome sequencing was
performed for clinical isolates using the hybrid strategy of combining Illumina
(short reads) and oxford nanopore (long reads) sequencing strategies. Clinical
isolates (n = 5) and vaccine strains (n = 7) genomes of B. pertussis from India
were compared with 744 B. pertussis closed genomes available in the public
databases. The phylogenomic comparison of B. pertussis genomes reported from India
will be advantageous in better understanding pertussis resurgence reported globally
with respect to pathogen adaptation. Department of Health Sciences, Symbiosis
International University, Pune 412115, India shweta.alai236@gmail.com;
m.gautam@seruminstitute.com; palkarsh@gmail.com; jsoswal@gmail.com;
sunil.gairola@seruminstitute.com; dhiraj.dhotre@nccs.res.in
10.3390/pathogens11070794 2022 11 7 - - 794 -
Fritsch, Hegger; Pereira, Felicidade; Costa, Erica; Fonseca, Vagner; Tosta,
Stephane; Xavier, Joilson; Levy, Flavia; Oliveira, Carla; Menezes, Gabriela; Lima,
Jaqueline; Santos, Lenisa; Silva, Luciana; Nardy, Vanessa; Astete, Marcela; Santos,
Beatriz; Aguiar, Nágila; Guedes, Maria; Faria, Guilherme; Furtini, Ronaldo;
Drumond, Safira; Cunha, Gabriel; Souza, Marcia; Jesus, Ronaldo; Guimarães, Sara;
Nuno, Italo; Santana, Ian; Sá, José; Santos, George; Silva, Willadesmon; Guedes,
Thiago; Araújo, Emerson; Said, Rodrigo; Albuquerque, Carlos; Peterka, Cassio;
Romano, Alessandro; Cunha, Rivaldo; Filippis, Ana; Leal e Silva de Mello, Arabela;
Giovanetti, Marta; Alcantara, Luiz Retrospective Investigation in Horses with
Encephalitis Reveals Unnoticed Circulation of West Nile Virus in Brazil
Viruses EN Brief Report WNV; nanopore sequencing; genomic
monitoring; Brazil During these past years, several studies have provided
serological evidence regarding the circulation of West Nile virus (WNV) in Brazil.
Despite some reports, much is still unknown regarding the genomic diversity and
transmission dynamics of this virus in the country. Recently, genomic monitoring
activities in horses revealed the circulation of WNV in several Brazilian regions.
These findings on the paucity of genomic data reinforce the need for prompt
investigation of WNV infection in horses, which may precede human cases of
encephalitis in Brazil. Thus, in this study, we retrospectively screened 54
suspicious WNV samples collected between 2017 and 2020 from the spinal cord and
brain of horses with encephalitis and generated three new WNV genomes from the
Ceará and Bahia states, located in the northeastern region of Brazil. The
Bayesian reconstruction revealed that at least two independent introduction events
occurred in Brazil. The first introduction event appears to be likely related to
the North American outbreak, and was estimated to have occurred in March 2013.The
second introduction event appears to have occurred in September 2017 and appears to
be likely related to the South American outbreak. Together, our results reinforce
the importance of increasing the priority of WNV genomic monitoring in equines with
encephalitis in order to track the dispersion of this emerging pathogen through the
country. Laboratorio de Pesquisa em Virologia Animal, Escola de Veterinária,
Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, Brazil
hegger.fritsch@gmail.com; felicidade.pereira@saude.ba.gov.br;
azevedoec@yahoo.com.br; vagner.fonseca@gmail.com; sttosta@gmail.com;
joilsonxavier@live.com; flaviallevy@yahoo.com.br; oliveirasc@yahoo.com.br;
gabrielaa.smenezes@gmail.com; jackgomes_@hotmail.com; dandarassa@hotmail.com;
roboredo.oliveira@gmail.com; vanessanardy@gmail.com; astetegomezmarcelak@gmail.com;
beatrizsenra.santos@gmail.com; naaguilar@hotmail.com; mariaisabel.guedes@gmail.com;
lsa@ima.mg.gov.br; ronaldoanas@ig.com.br; srmdrumond@hotmail.com;
cunha.gabrielmuricy@gmail.com; marciaspls@yahoo.com.br; ronaldo.jesus@saude.gov.br;
sarafrancoguimaraes@gmail.com; italoracema@outlook.com; ian_santana@outlook.com;
veterfarmac@gmail.com; georgedudananda@gmail.com;
willadesmon.silva@saude.ba.gov.br; thiago.guedes@saude.gov.br;
emerson.araujo@saude.gov.br; saidrod@paho.org; meloc@paho.org;
carlos.peterka@saude.gov.br; alessandropecego@gmail.com;
rivaldo.rivaldo.cunha@fiocruz.br; ana.bispo@ioc.fiocruz.br;
arabela.mello@saude.ba.gov.br; giovanetti.marta@gmail.com;
luiz.alcantara@ioc.fiocruz.br 10.3390/v14071540 2022 14 7 - - 1540
-
Zaitsev, Sergey; Khizhnyakova, Mariya; Feodorova, ValentinaRetrospective
Investigation of the Whole Genome of the Hypovirulent Listeria monocytogenes Strain
of ST201, CC69, Lineage III, Isolated from a Piglet with Fatal Neurolisteriosis
Microorganisms EN Communication Listeria monocytogenes;
neurolisteriosis; whole-genome sequencing (WGS); Oxford Nanopore; MLST; piglet;
ST201; CC69; lineage III; antimicrobial resistance; virulence-associated genes
Listeria monocytogenes (Lm), the causative agent for both human and animal
listeriosis, is considered to be a rare but potentially fatal foodborne pathogen.
While Lm strains associated with current cases of human listeriosis are now being
intensely investigated, our knowledge of this microorganism which has caused
listerial infection in the past is still extremely limited. The objective of this
study was a retrospective whole-genome sequence analysis of the Lm collection
strain, 4/52-1953, isolated in the middle of the 20th century from a piglet with
listerial neuroinfection. The multi-locus sequence typing (MLST) analysis based on
seven housekeeping genes (abcZ, bglA, cat, dapE, dat, ldh, and lhkA) showed that
the Lm strain 4/52-1953 was assigned to the sequence type 201 (ST201), clonal
complex 69 (CC69), and phylogenetic lineage III. The strain 4/52-1953, similarly to
other ST201 strains, probably originated from the ST9, CC69 via ST157. At least
eight different STs, ST69, ST72, ST130, ST136, ST148, ST469, ST769, and ST202, were
identified as the descendants of the first generation and a single one, ST2290, was
proved to be the descendant of the second generation. Among them there were strains
either associated with some sporadic cases of human and animal listerial infection
in the course of more than 60 years worldwide or isolated from food samples, fish
and dairy products, or migratory birds. Phylogenetic analysis based on whole
genomes of all the Lm strains available in the NCBI GenBank (n = 256) demonstrated
that the strain 4/52-1953 belonged to minor Cluster I, represented by lineage III
only, while two other major Clusters, II and III, were formed by lineages I and II.
In the genome of the strain 4/52-1953, 41 virulence-associated genes, including the
Listeria pathogenicity island 1 (LIPI-1), and LIPI-2 represented by two internalin
genes, the inlA and inlB genes, and five genes related to antibiotic resistance,
were found. These findings can help to make the emergence of both hyper- and
hypovirulent variants, including those bearing antibiotic resistance genes, more
visible and aid the aims of molecular epidemiology as well.Federal Research Center
for Virology and Microbiology, Branch in Saratov, 410028 Saratov, Russia
zaytsev-sergey@inbox.ru; khizhnyakova_mariya@mail.ru; feodorovav@mail.ru
10.3390/microorganisms10071442 2022 10 7 - - 1442 -
Paillet, Thomas; Lossouarn, Julien; Figueroa, Clarisse; Midoux, Cédric; Rué,
Olivier; Petit, Marie-Agnès; Dugat-Bony, Eric Virulent Phages Isolated from a
Smear-Ripened Cheese Are Also Detected in Reservoirs of the Cheese Factory
Viruses EN Article smear-ripened cheese; virulent phages; rind
bacteria; phage reservoirs; viral genomics Smear-ripened cheeses host complex
microbial communities that play a crucial role in the ripening process. Although
bacteriophages have been frequently isolated from dairy products, their diversity
and ecological role in such this type of cheese remain underexplored. In order to
fill this gap, the main objective of this study was to isolate and characterize
bacteriophages from the rind of a smear-ripened cheese. Thus, viral particles
extracted from the cheese rind were tested through a spot assay against a
collection of bacteria isolated from the same cheese and identified by sequencing
the full-length small subunit ribosomal RNA gene. In total, five virulent
bacteriophages infecting Brevibacterium aurantiacum, Glutamicibacter arilaitensis,
Leuconostoc falkenbergense and Psychrobacter aquimaris species were obtained. All
exhibit a narrow host range, being only able to infect a few cheese-rind isolates
within the same species. The complete genome of each phage was sequenced using both
Nanopore and Illumina technologies, assembled and annotated. A sequence comparison
with known phages revealed that four of them may represent at least new genera. The
distribution of the five virulent phages into the dairy-plant environment was also
investigated by PCR, and three potential reservoirs were identified. This work
provides new knowledge on the cheese rind viral community and an overview of the
distribution of phages within a cheese factory. Université Paris-Saclay, INRAE,
AgroParisTech, UMR SayFood, 91120 Palaiseau, France thomas.paillet@inrae.fr;
julien.lossouarn@inrae.fr; clarisse.figueroa4@gmail.com; cedric.midoux@inrae.fr;
olivier.rue@inrae.fr; marie-agnes.petit@inrae.fr; eric.dugat-bony@inrae.fr
10.3390/v14081620 2022 14 8 - - 1620 -
Li, Xiaoping; Kong, Ping; Daughtrey, Margery; Kosta, Kathleen; Schirmer, Scott;
Howle, Matthew; Likins, Michael; Hong, Chuanxue Characterization of the Soil
Bacterial Community from Selected Boxwood Gardens across the United States
Microorganisms EN Article disease suppressive soil; soil bacterial
community; urban garden; boxwood; biological control agents; Nanopore MinION
sequencing In a recent study, we observed a rapid decline of the boxwood blight
pathogen Calonectria pseudonaviculata (Cps) soil population in all surveyed gardens
across the United States, and we speculated that these garden soils might be
suppressive to Cps. This study aimed to characterize the soil bacterial community
in these boxwood gardens. Soil samples were taken from one garden in California,
Illinois, South Carolina, and Virginia and two in New York in early summer and late
fall of 2017 and 2018. Soil DNA was extracted and its 16S rRNA amplicons were
sequenced using the Nanopore MinION® platform. These garden soils were
consistently dominated by Rhizobiales and Burkholderiales, regardless of garden
location and sampling time. These two orders contain many species or strains
capable of pathogen suppression and plant fitness improvement. Overall, 66
bacterial taxa were identified in this study that are known to have strains with
biological control activity (BCA) against plant pathogens. Among the most abundant
were Pseudomonas spp. and Bacillus spp., which may have contributed to the Cps
decline in these garden soils. This study highlights the importance of soil
microorganisms in plant health and provides a new perspective on garden disease
management using the soil microbiome. Hampton Roads Agricultural Research and
Extension Center, Virginia Tech, Virginia Beach, VA 23455, USA lixiaopi@vt.edu;
pkong@vt.edu; mld9@cornell.edu; katfish@frontiernet.net;
scott.schirmer@illinois.gov; mhowle@clemson.edu; likinsm@chesterfield.gov;
chhong2@vt.edu 10.3390/microorganisms10081514 2022 10 8 - -
1514 -
Chen, Qiyan; Zou, Zhiyu; Cai, Chang; Li, Hui; Wang, Yang; Lei, Lei; Shao, Bing
Characterization of blaNDM-5-and blaCTX-M-199-Producing ST167 Escherichia
coli Isolated from Shared Bikes Antibiotics EN Article shared bikes; NDM-
5; CTX-M-199; ST167; whole genome analysis Shared bikes as a public transport
provide convenience for short-distance travel. Whilst they also act as a potential
vector for antimicrobial resistant (AR) bacteria and antimicrobial resistance genes
(ARGs). However, the understanding of the whole genome sequence of AR strains and
ARGs-carrying plasmids collected from shared bikes is still lacking. Here, we used
the HiSeq platform to sequence and analyze 24 Escherichia coli isolated from shared
bikes around Metro Stations in Beijing. The isolates from shared bikes showed 14
STs and various genotypes. Two blaNDM-5 and blaCTX-M-199-producing ST167 E. coli
have 16 resistance genes, four plasmid types and show >95% of similarities in
core genomes compared with the ST167 E. coli strains from different origins. The
blaNDM-5- or blaCTX-M-199-carrying plasmids sequencing by Nanopore were compared to
plasmids with blaNDM-5- or blaCTX-M-199 originated from humans and animals. These
two ST167 E. coli show high similarities in core genomes and the plasmid profiles
with strains from hospital inpatients and farm animals. Our study indicated that
ST167 E. coli is retained in diverse environments and carried with various
plasmids. The analysis of strains such as ST167 can provide useful information for
preventing or controlling the spread of AR bacteria between animals, humans and
environments. Beijing Key Laboratory of Detection Technology for Animal-Derived
Food Safety, College of Veterinary Medicine, China Agricultural University, Beijing
100193, China qiyanchen@cau.edu.cn; zouzhiyu@cau.edu.cn; c.cai@murdoch.edu.au;
lihui@bjcdc.org; wangyang@cau.edu.cn; leilei910@zafu.edu.cn; shaobingch@sina.com
10.3390/antibiotics11081030 2022 11 8 - - 1030 -
Khrenova, Maria; Panova, Tatiana; Rodin, Vladimir; Kryakvin, Maxim; Lukyanov,
Dmitrii; Osterman, Ilya; Zvereva, Maria Nanopore Sequencing for De Novo Bacterial
Genome Assembly and Search for Single-Nucleotide Polymorphism International
Journal of Molecular Sciences EN Article ONT sequencing; antibiotic
resistance; tolC gene; SNV; deletion Nanopore sequencing (ONT) is a new and
rapidly developing method for determining nucleotide sequences in DNA and RNA. It
serves the ability to obtain long reads of thousands of nucleotides without
assembly and amplification during sequencing compared to next-generation
sequencing. Nanopore sequencing can help for determination of genetic changes
leading to antibiotics resistance. This study presents the application of ONT
technology in the assembly of an E. coli genome characterized by a deletion of the
tolC gene and known single-nucleotide variations leading to antibiotic resistance,
in the absence of a reference genome. We performed benchmark studies to determine
minimum coverage depth to obtain a complete genome, depending on the quality of the
ONT data. A comparison of existing programs was carried out. It was shown that the
Flye program demonstrates plausible assembly results relative to others (Shasta,
Canu, and Necat). The required coverage depth for successful assembly strongly
depends on the size of reads. When using high-quality samples with an average read
length of 8 Kbp or more, the coverage depth of 30× is sufficient to assemble
the complete genome de novo and reliably determine single-nucleotide variations in
it. For samples with shorter reads with mean lengths of 2 Kbp, a higher coverage
depth of 50× is required. Avoiding of mechanical mixing is obligatory for
samples preparation. Nanopore sequencing can be used alone to determine
antibiotics-resistant genetic features of bacterial strains. Department of
Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia
mkhrenova@lcc.chem.msu.ru; tvk@genebee.msu.ru; rodinva@my.msu.ru;
maxim.kryakvin@gmail.com; dmitrii.lukianov@skoltech.ru; i.osterman@skoltech.ru;
zvereva@genebee.msu.ru 10.3390/ijms23158569 2022 23 15 - - 8569
-
Hu, Tingli; Chen, Guotao; Xu, Zhen; Luo, Site; Wang, Hui; Li, Chunlin; Shan, Lei;
Zhang, Baowei De Novo Whole-Genome Sequencing and Assembly of the Yellow-
Throated Bunting (Emberiza elegans) Provides Insights into Its Evolutionary
Adaptation Animals EN Article adaptation; Emberiza elegans; genome;
Nanopore sequencing Yellow-throated bunting is a small migratory songbird
unique to the Palearctic region. However, the genetic studies of this species
remain limited, with no nuclear genomic sequence reported to date. In this study,
the genomic DNA from the bird was sequenced in long reads using Nanopore sequencing
technology. Combining short-read sequencing, the genome was well-assembled and
annotated. The final length of the assembly is approximately 1.14 Gb, with a
scaffold N50 of 28.94 Mb. About 15,868 protein-coding genes were predicted, and
16.62% of the genome was identified as having repetitive elements. Comparative
genomic analysis showed numerous expanded gene families and positively selected
genes significantly enriched in those KEGG pathways that are associated with
migratory behavior adaptation and immune response. Here, this newly generated de
novo genome of the yellow-throated bunting using long reads provide the research
community with a valuable resource for further studies of population genetic
diversity and genome evolution in this species. School of Life Sciences, Anhui
University, Hefei 230601, China htl961029@163.com; 13865342219@163.com;
xuzhen0013@163.com; lstxmu@gmail.com; kikihui860425@163.com; lichunlin1985@163.com;
shanlei@njnu.edu.cn; zhangbw@ahu.edu.cn 10.3390/ani12152004 2022 12 15
- - 2004 -
do Amaral, Renan; Cardozo, Marita; Varani, Alessandro; Furquim, Maria; Dias, Clara;
Assis, William; da Silva, Alanderson; Herrera, Heitor; Machado, Rosangela; André,
Marcos First Report of Bartonella spp. in Marsupials from Brazil, with a
Description of Bartonella harrusi sp. nov. and a New Proposal for the Taxonomic
Reclassification of Species of the Genus Bartonella Microorganisms EN
Article bartonelosis; marsupialia; biochemical characterization;
phylogenomics; WGS; taxonomic classification The genus Bartonella (Rhizobiales:
Bartonellaceae) encompasses facultative intracellular Gram-negative
alphaproteobacteria that parasitize mainly erythrocytes and endothelial cells, as
well as macrophages, monocytes and dendritic cells. Although they can infect
numerous mammal species and arthropod vectors worldwide, reports of Bartonella
infections in marsupials are scarce. In fact, such agents have only been detected
in marsupials and/or associated ectoparasites in Australia and the United States of
America until the present moment. The present study aimed to isolate and
characterize molecularly, morphologically and phenotypically Bartonella infecting
free-living marsupials sampled in the Brazilian Pantanal, the largest wetland in
South America. Two marsupials were captured in December 2018 and six marsupials in
February 2019, totaling eight small mammals sampled: five (62.5%) Thylamys macrurus
and three (37.5%) Monodelphis domestica. All blood samples were submitted to qPCR
for Bartonella spp. based on the nuoG gene, a pre-enrichment liquid culture and a
chocolate agar solid culture. Bartonella sp. was isolated from 3 T. macrurus and
one M. domestica. One Bartonella isolate obtained from a T. macrurus blood sample
(strain 117A) that showed to be closely related to the Bartonella vinsonii complex
and Bartonella machadoae was selected for whole genome sequencing using a hybrid
approach based on Illumina NovaSeq and Nanopore sequencing platforms. This strain
showed a genome of 2.35 Mbp, with an average C + G content of 38.8%, coding for
2013 genes, and a 29 kb plasmid with an average C + G content of 34.5%. In
addition, this strain exhibited an average nucleotide identity (ANI) of 85% with
Bartonella species belonging to the B. vinsonii group and 91% with B. machadoae.
Phylogenomic analysis based on 291 protein coding genes shared by the genomes of 53
Bartonella species positioned this strain closely to B. machadoae. This new
isolated species was named Bartonella harrusi sp. nov., which was characterized as
having small capnophilic, microaerophilic and aerobic rods with an absence of pili
and flagella. In conclusion, the present work describes the biochemical, phenotypic
and genomic characteristics of Bartonella harrusi, a new species isolated from the
T. macrurus blood samples of the Brazilian Pantanal. Finally, a review of the
taxonomic classification of members of the genus Bartonella is proposed, based on
the ANI values accessed by whole genome sequencing analyses. Programa de Pós-
Graduação em Microbiologia Agropecuária, Faculdade de Ciências Agrárias e
Veterinárias, Universidade Estadual Paulista (UNESP), Jaboticabal 15385-000, SP,
Brazil renan.amaral@unesp.br; marita.vedovelli@unesp.br;
alessandro.varani@unesp.br; mecfurquim@yahoo.com.br; clara.morato@unesp.br;
william.oliveira.assis@gmail.com; alander_rodrigue@hotmail.com; herrera@ucdb.br;
rzacariasmachado@gmail.com; mr.andre@unesp.br 10.3390/microorganisms10081609
2022 10 8 - - 1609 -
Kirov, Ilya; Kolganova, Elizaveta; Dudnikov, Maxim; Yurkevich, Olga; Amosova,
Alexandra; Muravenko, Olga A Pipeline NanoTRF as a New Tool for De Novo
Satellite DNA Identification in the Raw Nanopore Sequencing Reads of Plant Genomes
Plants EN Article satellite DNA; Nanopore sequencing; genome;
tandem repeats; pipeline High-copy tandemly organized repeats (TRs), or
satellite DNA, is an important but still enigmatic component of eukaryotic genomes.
TRs comprise arrays of multi-copy and highly similar tandem repeats, which makes
the elucidation of TRs a very challenging task. Oxford Nanopore sequencing data
provide a valuable source of information on TR organization at the single molecule
level. However, bioinformatics tools for de novo identification of TRs in raw
Nanopore data have not been reported so far. We developed NanoTRF, a new python
pipeline for TR repeat identification, characterization and consensus monomer
sequence assembly. This new pipeline requires only a raw Nanopore read file from
low-depth (<1×) genome sequencing. The program generates an informative
html report and figures on TR genome abundance, monomer sequence and monomer
length. In addition, NanoTRF performs annotation of transposable elements (TEs)
sequences within or near satDNA arrays, and the information can be used to
elucidate how TR–TE co-evolve in the genome. Moreover, we validated by FISH
that the NanoTRF report is useful for the evaluation of TR chromosome
organization—clustered or dispersed. Our findings showed that NanoTRF is a
robust method for the de novo identification of satellite repeats in raw Nanopore
data without prior read assembly. The obtained sequences can be used in many
downstream analyses including genome assembly assistance and gap estimation,
chromosome mapping and cytogenetic marker development. All-Russia Research
Institute of Agricultural Biotechnology, Timiryazevskaya Str. 42, Moscow 127550,
Russia kirovez@gmail.com; liza.colg@gmail.com; max.dudnikov.07@gmail.com;
olikys@gmail.com; amomar@mail.ru; olgmur1@yandex.ru 10.3390/plants11162103 2022
11 16 - - 2103 -
Werner, David; Acharya, Kishor; Blackburn, Adrian; Zan, Rixia; Plaimart, Jidapa;
Allen, Ben; Mgana, Shaaban; Sabai, Shadrack; Halla, Franella; Massawa, Said; Haile,
Alemseged; Hiruy, Andualem; Mohammed, Jemila; Vinitnantharat, Soydoa; Thongsamer,
Thunchanok; Pantha, Kalyan; Mota Filho, Cesar; Lopes, BrunaMinION Nanopore
Sequencing Accelerates Progress towards Ubiquitous Genetics in Water Research
Water EN Review MinION; nanopore sequencing; NGS; eDNA; water
research; ubiquitous genetics; SDG6 In 2014, Oxford Nanopore Technologies (ONT)
introduced an affordable and portable sequencer called MinION. We reviewed emerging
applications in water research and assessed progress made with this platform
towards ubiquitous genetics. With >99% savings in upfront costs as compared to
conventional platforms, the MinION put sequencing capacity into the hands of many
researchers and enabled novel applications with diverse remits, including in
countries without universal access to safe water and sanitation. However, to
realize the MinION’s fabled portability, all the auxiliary equipment items
for biomass concentration, genetic material extraction, cleanup, quantification,
and sequencing library preparation also need to be lightweight and affordable. Only
a few studies demonstrated fully portable workflows by using the MinION onboard a
diving vessel, an oceanographic research ship, and at sewage treatment works. Lower
nanopore sequencing read accuracy as compared to alternative platforms currently
hinders MinION applications beyond research, and inclusion of positive and negative
controls should become standard practice. ONT’s EPI2ME platform is a major
step towards user-friendly bioinformatics. However, no consensus has yet emerged
regarding the most appropriate bioinformatic pipeline, which hinders
intercomparison of study results. Processing, storing, and interpreting large data
sets remains a major challenge for ubiquitous genetics and democratizing sequencing
applications. School of Engineering, Newcastle University, Newcastle upon Tyne
NE1 7RU, UK david.werner@newcastle.ac.uk; kishor.acharya@newcastle.ac.uk;
adrian.blackburn@newcastle.ac.uk; r.zan2@newcastle.ac.uk;
j.plaimart2@newcastle.ac.uk; ben.allen@newcastle.ac.uk; smmgana@gmail.com;
sabaismm@gmail.com; frannyhalla@yahoo.com; manenosaid@gmail.com;
a.t.haile@cgiar.org; andumk21@gmail.com; emuamarj.j89@gmail.com;
soydoa.vin@mail.kmutt.ac.th; thunchanok.th@mail.kmutt.ac.th;
pantha.kalyan@gmail.com; crmota@gmail.com; bruna.coelho.lopes@gmail.com
10.3390/w14162491 2022 14 16 - - 2491 -
Marin, Clara; Marco-Jiménez, Francisco; Martínez-Priego, Llucia; De Marco-Romero,
Griselda; Soriano-Chirona, Vicente; Lorenzo-Rebenaque, Laura; D’Auria, Giuseppe
Rapid Oxford Nanopore Technologies MinION Sequencing Workflow for
Campylobacter jejuni Identification in Broilers on Site—A Proof-of-
Concept Study Animals EN Article poultry; foodborne; 16S RNA;
microbiota; Bento Lab Campylobacter is recognised as one of the most important
foodborne bacteria, with a worldwide health and socioeconomic impact. This
bacterium is one of the most important zoonotic players in poultry, where efficient
and fast detection methods are required. Current official culture methods for
Campylobacter enumeration in poultry usually include >44 h of culture and >72
h for identification, thus requiring at least five working shifts (ISO/TS 10272-
2:2017). Here, we have assembled a portable sequencing kit composed of the Bento
Lab and the MinION and developed a workflow for on-site farm use that is able to
detect and report the presence of Campylobacter from caecal samples in less than
five hours from sampling time, as well as the relationship of Campylobacter with
other caecal microbes. Beyond that, our workflow may offer a cost-effective and
practical method of microbiologically monitoring poultry at the farm. These results
would demonstrate the possibility of carrying out rapid on-site screening to
monitor the health status of the poultry farm/flock during the production chain.
Institute of Biomedical Sciences, Veterinary Faculty, Universidad Cardenal
Herrera-CEU, 46113 Alfara del Patriarca, Spain clara.marin@uchceu.es;
fmarco@dca.upv.es; martinez_lucpri@gva.es; demarco_gri@gva.es;
soriano_vicchi@gva.es; laura.lorenzorebenaque@uchceu.es; dauria_giu@gva.es
10.3390/ani12162065 2022 12 16 - - 2065 -
Schafhauser, Thomas; Wibberg, Daniel; Binder, Antonia; Rückert, Christian; Busche,
Tobias; Wohlleben, Wolfgang; Kalinowski, Jörn Genome Assembly and Genetic Traits
of the Pleuromutilin-Producer Clitopilus passeckerianus DSM1602 Journal of Fungi
EN Article genome assembly; Nanopore sequencing; Illumina sequencing;
Clitopilus; Agaricales; mitochondrial genome; heteroplasmic mycelium;
pleuromutilin; terpene synthase; biosynthetic gene cluster The gilled mushroom
Clitopilus passeckerianus (Entolomataceae, Agaricales, Basidiomycota) is well known
to produce the terpenoid pleuromutilin, which is the biotechnological basis for
medically important antibiotics such as lefamulin and retapamulin. Their unique
mode of action and good tolerance entails an increasing demand of pleuromutilin-
derived antibiotics in veterinary and human health care. Surprisingly, despite
their pharmaceutical importance, no genome sequence is available of any
pleuromutilin-producing fungus. Here, we present the high-quality draft genome
sequence of the pleuromutilin-producer C. passeckerianus DSM1602 including
functional genome annotation. More precisely, we employed a hybrid assembly
strategy combining Illumina sequencing and Nanopore sequencing to assemble the
mitochondrial genome as well as the nuclear genome. In accordance with the
dikaryotic state of the fungus, the nuclear genome has a diploid character.
Interestingly, the mitochondrial genome appears duplicated. Bioinformatic analysis
revealed a versatile secondary metabolism with an emphasis on terpenoid
biosynthetic enzymes in C. passeckerianus and also in related strains. Two alleles
of biosynthetic gene clusters for pleuromutilin were found in the genome of C.
passeckerianus. The pleuromutilin genes were reassembled with yeast-specific
elements for heterologous expression in Saccharomyces cerevisiae. Our work lays the
foundation for metabolic strain engineering towards higher yields of the valuable
compound pleuromutilin. Mikrobiologie und Biotechnologie, Interfakultäres Institut
für Mikrobiologie und Infektionsmedizin, Eberhard Karls Universität Tübingen, Auf
der Morgenstelle 28, 72076 Tuebingen, Germany thomas.schafhauser@biotech.uni-
tuebingen.de; dwibberg@cebitec.uni-bielefeld.de; ant.binder@web.de;
christian.rueckert@cebitec.uni-bielefeld.de; tbusche@iit-biotech.de;
wolfgang.wohlleben@biotech.uni-tuebingen.de; joern@cebitec.uni-bielefeld.de
10.3390/jof8080862 2022 8 8 - - 862 -
Xiaokaiti, Xiakena; Hashiguchi, Yasuyuki; Ota, Hidetoshi; Kumazawa, Yoshinori
Evolution of the Noncoding Features of Sea Snake Mitochondrial Genomes within
Elapidae Genes EN Article control region; tandem repeat; Nanopore
sequencing; light-strand replication origin Mitochondrial genomes of four
elapid snakes (three marine species [Emydocephalus ijimae, Hydrophis ornatus, and
Hydrophis melanocephalus], and one terrestrial species [Sinomicrurus japonicus])
were completely sequenced by a combination of Sanger sequencing, next-generation
sequencing and Nanopore sequencing. Nanopore sequencing was especially effective in
accurately reading through long tandem repeats in these genomes. This led us to
show that major noncoding regions in the mitochondrial genomes of those three sea
snakes contain considerably long tandem duplications, unlike the mitochondrial
genomes previously reported for same and other sea snake species. We also found a
transposition of the light-strand replication origin within a tRNA gene cluster for
the three sea snakes. This change can be explained by the Tandem
Duplication—Random Loss model, which was further supported by remnant
intervening sequences between tRNA genes. Mitochondrial genomes of true snakes
(Alethinophidia) have been shown to contain duplicate major noncoding regions, each
of which includes the control region necessary for regulating the heavy-strand
replication and transcription from both strands. However, the control region
completely disappeared from one of the two major noncoding regions for two
Hydrophis sea snakes, posing evolutionary questions on the roles of duplicate
control regions in snake mitochondrial genomes. The timing and molecular mechanisms
for these changes are discussed based on the elapid phylogeny. Department of
Information and Basic Science and Research Center for Biological Diversity,
Graduate School of Science, Nagoya City University, Nagoya 467-8501, Japan
xiakena@nsc.nagoya-cu.ac.jp; yasuyuki.hashiguchi@ompu.ac.jp;
ohta@hitohaku.jp; kuma@nsc.nagoya-cu.ac.jp 10.3390/genes13081470 2022 13
8 - - 1470 -
Israeli, Ofir; Guedj-Dana, Yehoudit; Shifman, Ohad; Lazar, Shirley; Cohen-Gihon,
Inbar; Amit, Sharon; Ben-Ami, Ronen; Paran, Nir; Schuster, Ofir; Weiss, Shay; Zvi,
Anat; Beth-Din, Adi Rapid Amplicon Nanopore Sequencing (RANS) for the
Differential Diagnosis of Monkeypox Virus and Other Vesicle-Forming Pathogens
Viruses EN Communication Monkeypox virus; Oxford nanopore;
Flongle; Orthopoxvirus; vesicle-forming pathogens; variola virus; smallpox As of
July 2022, more than 16,000 laboratory-confirmed monkeypox (MPX) cases have been
reported worldwide. Until recently, MPX was a rare viral disease seldom detected
outside Africa. MPX virus (MPXV) belongs to the Orthopoxvirus (OPV) genus and is a
genetically close relative of the Variola virus (the causative agent of smallpox).
Following the eradication of smallpox, there was a significant decrease in
smallpox-related morbidity and the population’s immunity to other OPV-related
diseases such as MPX. In parallel, there was a need for differential diagnosis
between the different OPVs’ clinical manifestations and diseases with similar
symptoms (i.e., chickenpox, herpes simplex). The current study aimed to provide a
rapid genetic-based diagnostic tool for accurate and specific identification of
MPXV and additional related vesicle-forming pathogens. We initially assembled a
list of 14 relevant viral pathogens, causing infectious diseases associated with
vesicles, prone to be misdiagnosed as MPX. Next, we developed an approach that we
termed rapid amplicon nanopore sequencing (RANS). The RANS approach uses diagnostic
regions that harbor high homology in their boundaries and internal diagnostic SNPs
that, when sequenced, aid the discrimination of each pathogen within a group.
During a multiplex PCR amplification, a dA tail and a 5′-phosphonate were
simultaneously added, thus making the PCR product ligation ready for nanopore
sequencing. Following rapid sequencing (a few minutes), the reads were compared to
a reference database and the nearest strain was identified. We first tested our
approach using samples of known viruses cultured in cell lines. All the samples
were identified correctly and swiftly. Next, we examined a variety of clinical
samples from the 2022 MPX outbreak. Our RANS approach identified correctly all the
PCR-positive MPXV samples and mapped them to strains that were sequenced during the
2022 outbreak. For the subset of samples that were negative for MPXV by PCR, we
obtained definite results, identifying other vesicle-forming viruses: Human
herpesvirus 3, Human herpesvirus 2, and Molluscum contagiosum virus. This work was
a proof-of-concept study, demonstrating the potential of the RANS approach for
rapid and discriminatory identification of a panel of closely related pathogens.
The simplicity and affordability of our approach makes it straightforward to
implement in any genetics lab. Moreover, other differential diagnostics panels
might benefit from the implementation of the RANS approach into their diagnostics
pipelines. Departments of Biochemistry and Molecular Genetics, Israel Institute
for Biological Research (IIBR), Ness Ziona 74100, Israel ofiri@iibr.gov.il;
yehouditg@iibr.gov.il; ohads@iibr.gov.il; shirleyl@iibr.gov.il; inbarg@iibr.gov.il;
sharon.amit@sheba.health.gov.il; ronenba@tlvmc.gov.il; nirp@iibr.gov.il;
ofirsc@iibr.gov.il; shayw@iibr.gov.il; anatz@iibr.gov.il; adib@iibr.gov.il
10.3390/v14081817 2022 14 8 - - 1817 -
Hu, Wenjie; Zhang, Yuxin; Zhang, Hongrui; Chen, Weigang Hardware Acceleration of
Identifying Barcodes in Multiplexed Nanopore Sequencing Electronics EN
Article multiplexed sequencing; DNA sequencing barcode; dynamic
programming; FPGA In multiplexed sequencing, the identification of DNA sequencing
barcodes can effectively reduce the probability of sample misassignment. However,
the great quantity of sequence data requires a high-throughput identification
method. Therefore, based on a barcode identification scheme combining cyclic
shifting with dynamic programming (DP), this paper proposes, implements and tests a
hardware accelerator that can accelerate barcode identification. In the
accelerator, considering that the computational complexity of the DP algorithm can
be expressed as the multiplication of the lengths of both involved sequences, we
design a systolic array structure with simplified processing element (PE) and a
parallel circuit architecture to identify the insertion and deletion errors based
on the traceback. The accelerator is implemented on a field-programmable gate array
(FPGA), and its performance is compared with that of software implemented on a
general-purpose computer. The experimental results indicate that, compared with the
software implementation, the accelerator can achieve speedups of two orders of
magnitude for longer barcodes. School of Microelectronics, Tianjin University,
Tianjin 300072, China 2019232176@tju.edu.cn; zhangyx2020@tju.edu.cn;
3018205104@tju.edu.cn; chenwg@tju.edu.cn 10.3390/electronics11162596 2022 11
16 - - 2596 -
Mgwatyu, Yamkela; Cornelissen, Stephanie; van Heusden, Peter; Stander, Allison;
Ranketse, Mary; Hesse, Uljana Establishing MinION Sequencing and Genome Assembly
Procedures for the Analysis of the Rooibos (Aspalathus linearis) Genome Plants
EN Article rooibos; plant genome assembly; Oxford Nanopore; Canu;
MaSuRCA; Haslr; Wengan; Flye; Redbean; Raven; NextDenovo; Racon; Medaka; Nextpolish
While plant genome analysis is gaining speed worldwide, few plant genomes
have been sequenced and analyzed on the African continent. Yet, this information
holds the potential to transform diverse industries as it unlocks medicinally and
industrially relevant biosynthesis pathways for bioprospecting. Considering that
South Africa is home to the highly diverse Cape Floristic Region, local
establishment of methods for plant genome analysis is essential. Long-read
sequencing is becoming standard procedure for plant genome research, as these reads
can span repetitive regions of the DNA, substantially facilitating reassembly of a
contiguous genome. With the MinION, Oxford Nanopore offers a cost-efficient
sequencing method to generate long reads; however, DNA purification protocols must
be adapted for each plant species to generate ultra-pure DNA, essential for these
analyses. Here, we describe a cost-effective procedure for the extraction and
purification of plant DNA and evaluate diverse genome assembly approaches for the
reconstruction of the genome of rooibos (Aspalathus linearis), an endemic South
African medicinal plant widely used for tea production. We discuss the pros and
cons of nine tested assembly programs, specifically Redbean and NextDenovo, which
generated the most contiguous assemblies, and Flye, which produced an assembly
closest to the predicted genome size. Department of Biotechnology, University
of the Western Cape, Robert Sobukwe Road, Bellville 7535, South Africa
yamkelamgwatyu@gmail.com; stephcor11@gmail.com; pvh@sanbi.ac.za;
allison.stander@outlook.com; mranketse@gmail.com; uhesse@uwc.ac.za
10.3390/plants11162156 2022 11 16 - - 2156 -
Ogaji, Yvonne; Lee, Robert; Sawbridge, Tim; Cocks, Benjamin; Daetwyler, Hans; Kaur,
Sukhjiwan De Novo Long-Read Whole-Genome Assemblies and the Comparative Pan-
Genome Analysis of Ascochyta Blight Pathogens Affecting Field Pea Journal of
Fungi EN Article nuclear genome; mitochondrial genome; CAZymes; orthologs;
comparative genomics Ascochyta Blight (AB) is a major disease of many cool-
season legumes globally. In field pea, three fungal pathogens have been identified
to be responsible for this disease in Australia, namely Peyronellaea pinodes,
Peyronellaea pinodella and Phoma koolunga. Limited genomic resources for these
pathogens have been generated, which has hampered the implementation of effective
management strategies and breeding for resistant cultivars. Using Oxford Nanopore
long-read sequencing, we report the first high-quality, fully annotated, near-
chromosome-level nuclear and mitochondrial genome assemblies for 18 isolates from
the Australian AB complex. Comparative genome analysis was performed to elucidate
the differences and similarities between species and isolates using phylogenetic
relationships and functional diversity. Our data indicated that P. pinodella and P.
koolunga are heterothallic, while P. pinodes is homothallic. More homology and
orthologous gene clusters are shared between P. pinodes and P. pinodella compared
to P. koolunga. The analysis of the repetitive DNA content showed differences in
the transposable repeat composition in the genomes and their expression in the
transcriptomes. Significant repeat expansion in P. koolunga’s genome was
seen, with strong repeat-induced point mutation (RIP) activity being evident.
Phylogenetic analysis revealed that genetic diversity can be exploited for species
marker development. This study provided the much-needed genetic resources and
characterization of the AB species to further drive research in key areas such as
disease epidemiology and host–pathogen interactions. Agriculture Victoria,
AgriBio, Centre for AgriBioscience, 5 Ring Road, Melbourne, VIC 3083, Australia
yvonne.ogaji@agriculture.vic.gov.au; robert.lee@curtin.edu.au;
tim.sawbridge@agriculture.vic.gov.au; ben.cocks@agriculture.vic.gov.au;
hansdd@gmail.com; sukhjiwan.kaur@agriculture.vic.gov.au 10.3390/jof8080884
2022 8 8 - - 884 -
Esnault, Gaelle; Earley, Bernadette; Cormican, Paul; Waters, Sinead; Lemon, Ken;
Cosby, S.; Lagan, Paula; Barry, Thomas; Reddington, Kate; McCabe, Matthew
Assessment of Rapid MinION Nanopore DNA Virus Meta-Genomics Using Calves
Experimentally Infected with Bovine Herpes Virus-1 Viruses EN Article
Oxford Nanopore Technologies; MinION; Epi2ME; rapid viral metagenomics
diagnostics; bovine herpesvirus 1; bovine respiratory disease Bovine respiratory
disease (BRD), which is the leading cause of morbidity and mortality in cattle, is
caused by numerous known and unknown viruses and is responsible for the widespread
use of broad-spectrum antibiotics despite the use of polymicrobial BRD vaccines.
Viral metagenomics sequencing on the portable, inexpensive Oxford Nanopore
Technologies MinION sequencer and sequence analysis with its associated user-
friendly point-and-click Epi2ME cloud-based pathogen identification software has
the potential for point-of-care/same-day/sample-to-result metagenomic sequence
diagnostics of known and unknown BRD pathogens to inform a rapid response and
vaccine design. We assessed this potential using in vitro viral cell cultures and
nasal swabs taken from calves that were experimentally challenged with a single
known BRD-associated DNA virus, namely, bovine herpes virus 1. Extensive
optimisation of the standard Oxford Nanopore library preparation protocols,
particularly a reduction in the PCR bias of library amplification, was required
before BoHV-1 could be identified as the main virus in the in vitro cell cultures
and nasal swab samples within approximately 7 h from sample to result. In addition,
we observed incorrect assignment of the bovine sequence to bacterial and viral taxa
due to the presence of poor-quality bacterial and viral genome assemblies in the
RefSeq database used by the EpiME Fastq WIMP pathogen identification software.
Animal and Bioscience Research Department, Animal & Grassland Research
and Innovation Centre, Teagasc, Oak Park, R93 XE12 Carlow, Ireland
gaelle.esnault@gmail.com; bernadette.earley@teagasc.ie;
paul.cormican@teagasc.ie; sinead.waters@teagasc.ie; kenneth.lemon@afbini.gov.uk;
louise.cosby@afbini.gov.uk; paula.lagan@afbini.gov.uk; thomas.barry@nuigalway.ie;
kate.reddington@nuigalway.ie; matthew.mccabe@teagasc.ie 10.3390/v14091859 2022
14 9 - - 1859 -
Feng, Yin-Chih; Liou, Ci-Hong; Ng, Wailap; Chen, Feng-Jui; Hung, Chih-Hsin; Liu,
Po-Yen; Liao, Yu-Chieh; Wu, Han-Chieh; Cheng, Ming-Fang Distribution and Genomic
Characterization of Third-Generation Cephalosporin-Resistant Escherichia coli
Isolated from a Single Family and Home Environment: A 2-Year Longitudinal Study
Antibiotics EN Article Escherichia coli; third-generation
cephalosporin-resistant Escherichia coli; extended-spectrum β-lactamases; plasmid;
AmpC β-lactamases; CMY-2; whole-genome sequencing Third-generation
cephalosporin-resistant Escherichia coli (CREC), particularly strains producing
extended-spectrum β-lactamases (ESBLs), are a global concern. Our study aims
to longitudinally assemble the genomic characteristics of CREC isolates from fecal
samples from an index patient with recurrent CREC-related urinary tract infections
and his family and swabs from his home environment 12 times between 2019 and 2021
to investigate the distribution of antibiotic resistance genes. CREC identified
using the VITEK 2 were subjected to nanopore whole-genome sequencing (WGS). The WGS
of 27 CREC isolates discovered in 137 specimens (1 urine, 123 feces, and 13
environmental) revealed the predominance of ST101 and ST131. Among these sequence
types, blaCTX-M (44.4%, n = 12) was the predominant ESBL gene family, with blaCTX-
M-14 (n = 6) being the most common. The remaining 15 (55.6%) isolates harbored
blaCMY-2 genes and were clonally diverse. All E. coli isolated from the index
patient’s initial urine and fecal samples belonged to O25b:H4-B2-ST131 and
carried blaCTX-M-14. The results of sequence analysis indicate plasmid-mediated
household transmission of blaCMY-2 or blaCTX-M-55. A strong genomic similarity was
discovered between fecal ESBL-producing E. coli and uropathogenic strains.
Furthermore, blaCMY-2 genes were widely distributed among the CREC isolated from
family members and their home environment. Department of Pediatrics, Kaohsiung
Veterans General Hospital, Kaohsiung 813414, Taiwan simp60128@yahoo.com.tw;
cihongliou@nhri.edu.tw; wailap.ng@gmail.com; frchen@nhri.edu.tw; chhung@isu.edu.tw;
pyliu@vghks.gov.tw; jade@nhri.org.tw; hanjie@nhri.edu.tw; mfcheng@vghks.gov.tw
10.3390/antibiotics11091152 2022 11 9 - - 1152 -
Lu, Ruisen; Liu, Jia; Wang, Xuegang; Song, Zhao; Ji, Xiangdong; Li, Naiwei; Ma,
Gang; Sun, Xiaoqin Chromosome-Level Genome Assembly of a Fragrant Japonica
Rice Cultivar ‘Changxianggeng 1813’ Provides Insights into Genomic
Variations between Fragrant and Non-Fragrant Japonica Rice International Journal of
Molecular Sciences EN Article BADH2; ‘Changxianggeng 1813’; fragrant
rice; genome assembly; genomic variations; japonica cultivar East Asia has an
abundant resource of fragrant japonica rice that is gaining increasing interest
among both consumers and producers. However, genomic resources and in particular
complete genome sequences currently available for the breeding of fragrant japonica
rice are still scarce. Here, integrating Nanopore long-read sequencing, Illumina
short-read sequencing, and Hi-C methods, we presented a high-quality chromosome-
level genome assembly (~378.78 Mb) for a new fragrant japonica cultivar
‘Changxianggeng 1813’, with 31,671 predicated protein-coding genes.
Based on the annotated genome sequence, we demonstrated that it was the badh2-E2
type of deletion (a 7-bp deletion in the second exon) that caused fragrance in
‘Changxianggeng 1813’. Comparative genomic analyses revealed that
multiple gene families involved in the abiotic stress response were expanded in the
‘Changxianggeng 1813’ genome, which further supported the previous
finding that no generalized loss of abiotic stress tolerance associated with the
fragrance phenotype. Although the ‘Changxianggeng 1813’ genome showed
high genomic synteny with the genome of the non-fragrant japonica rice cultivar
Nipponbare, a total of 289,970 single nucleotide polymorphisms (SNPs), 96,093 small
insertion-deletion polymorphisms (InDels), and 8690 large structure variants (SVs,
>1000 bp) were identified between them. Together, these genomic resources will
be valuable for elucidating the mechanisms underlying economically important traits
and have wide-ranging implications for genomics-assisted breeding in fragrant
japonica rice. Institute of Botany, Jiangsu Province and Chinese Academy of
Sciences, Nanjing 210014, China lurs@cnbg.net; liujia@cnbg.net; wlzh21@163.com;
songzhao@zju.edu.cn; jixiangdong0324@sina.com; linaiwei@jib.ac.cn;
changshumagang@163.com; xiaoqinsun@cnbg.net 10.3390/ijms23179705 2022 23
17 - - 9705 -
Madi, Nada; Sadeq, Mohammad; Essa, Sahar; Safar, Hussain; Al-Adwani, Anfal; Al-
Khabbaz, Marwa Strain Variation Based on Spike Glycoprotein Gene of SARS-CoV-2
in Kuwait from 2020 to 2021 Pathogens EN Article SARS-CoV-2; spike
glycoprotein; strain variation; Kuwait Severe acute respiratory syndrome
coronavirus-2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019
(COVID-19), which was first identified in Wuhan, China, in December 2019. With the
global transmission of the virus, many SARS-CoV-2 variants have emerged due to the
alterations of the spike glycoprotein. Therefore, the S glycoprotein encoding gene
has widely been used for the molecular analysis of SARS-Co-2 due to its features
affecting antigenicity and immunogenicity. We analyzed the S gene sequences of 35
SARS-CoV-2 isolates in Kuwait from March 2020 to February 2021 using the Sanger
method and MinION nanopore technology to confirm novel nucleotide alterations. Our
results show that the Kuwaiti strains from clade 19A and B were the dominant
variants early in the pandemic, while clade 20I (Alpha, V1) was the dominant
variant from February 2021 onward. Besides the known mutations, 21 nucleotide
deletions in the S glycoprotein in one Kuwaiti strain were detected, which might
reveal a recombinant SARS-CoV-2 with the defective viral genome (DVG). This study
emphasizes the importance of closely perceiving the emerging clades with these
mutations during this continuous pandemic as some may influence the specificity of
diagnostic tests, such as RT-PCR and even vaccine design directing these positions.
Virology Unit, Department of Microbiology, Faculty of Medicine, Kuwait
University, Safat 13110, Kuwait nada.madi@ku.edu.kw; dr.abul85@hotmail.com;
sahar.essa@ku.edu.kw; hussain.safar@ku.edu.kw; anfal.a@ku.edu.kw;
marwah.alkhabbaz@ku.edu.kw 10.3390/pathogens11090985 2022 11 9 -
- 985 -
Kasibut, Punnisa; Kuvatanasuchati, Jintakorn; Thaweboon, Boonyanit; Sirisoontorn,
Irin Oral Microbiome in Orthodontic Acrylic Retainer Polymers EN Article
oral microbiome; orthodontic; acrylic retainer The oral microbiome can be
shifted if the patients wear the acrylic retainers for a lengthy period. It is
essential to understand the components of the plaque in order to forestall the
development of dental caries and gingivitis. The aim of this study is to report the
bacterial communities that adhere to the acrylic retainers by full-length nanopore
16S sequencing. Six healthy participants were allocated into 2 groups (chemical
tablet and brushing groups). Plaque samples were collected from the acrylic
retainer surfaces before and after cleaning. The bacterial communities were
reported using full-length nanopore 16S sequencing. The results showed that 7
distinct phyla were identified by sequencing. The most prevalent of these was the
Firmicutes. We found a total of 72 genera. The most common microorganism across all
samples was Streptococcus, followed by Neisseria, Rothia, and Gemella. The beta
diversity showed a significant difference between before and after cleaning (p <
0.05). This study revealed the novel finding that a combination of chemical and
mechanical cleaning methods was the most effective method of eliminating retainer
biofilms. Moreover, retainer cleaning tablets did not alter the homeostatic balance
of the bacterial communities adhering to the acrylic retainers. Department of
Clinical Dentistry, Walailak University International College of Dentistry (WUICD),
87 Ranong 2 Road, Dusit, Bangkok 10300, Thailand punnisakasibut1@gmail.com;
jintakorn.ku@wu.ac.th; tboonit@gmail.com; irin.sirisoontorn@gmail.com
10.3390/polym14173583 2022 14 17 - - 3583 -
Helal, Asmaa; Saad, Bishoy; Saad, Mina; Mosaad, Gamal; Aboshanab, Khaled
Evaluation of the Available Variant Calling Tools for Oxford Nanopore
Sequencing in Breast Cancer Genes EN Article nanopore; variant detection;
human-SNP-wf; Clair3; Clair; NanoCaller; Longshot; Medaka The goal of biomarker
testing, in the field of personalized medicine, is to guide treatments to achieve
the best possible results for each patient. The accurate and reliable
identification of everyone’s genome variants is essential for the success of
clinical genomics, employing third-generation sequencing. Different variant calling
techniques have been used and recommended by both Oxford Nanopore Technologies
(ONT) and Nanopore communities. A thorough examination of the variant callers might
give critical guidance for third-generation sequencing-based clinical genomics. In
this study, two reference genome sample datasets (NA12878) and (NA24385) and the
set of high-confidence variant calls provided by the Genome in a Bottle (GIAB) were
used to allow the evaluation of the performance of six variant calling tools,
including Human-SNP-wf, Clair3, Clair, NanoCaller, Longshot, and Medaka, as an
integral step in the in-house variant detection workflow. Out of the six variant
callers understudy, Clair3 and Human-SNP-wf that has Clair3 incorporated into it
achieved the highest performance rates in comparison to the other variant callers.
Evaluation of the results for the tool was expressed in terms of Precision, Recall,
and F1-score using Hap.py tools for the comparison. In conclusion, our findings
give important insights for identifying accurate variants from third-generation
sequencing of personal genomes using different variant detection tools available
for long-read sequencing. Department of Bioinformatics, HITS Solutions Co.,
Cairo 11765, Egypt asmaaa@hitstechnology.com; bishoyth@hitssolutions.com;
minath@hitssolutions.com; shenouda@hitssolutions.com;
aboshanab2012@pharma.asu.edu.eg 10.3390/genes13091583 2022 13 9 -
- 1583 -
Quezada-Aguiluz, Mario; Opazo-Capurro, Andrés; Lincopan, Nilton; Esposito,
Fernanda; Fuga, Bruna; Mella-Montecino, Sergio; Riedel, Gisela; Lima, Celia; Bello-
Toledo, Helia; Cifuentes, Marcela; Silva-Ojeda, Francisco; Barrera, Boris;
Hormazábal, Juan; González-Rocha, Gerardo Novel Megaplasmid Driving NDM-1-Mediated
Carbapenem Resistance in Klebsiella pneumoniae ST1588 in South America Antibiotics
EN Brief Report NDM-1; carbapenem-resistant Enterobacterales;
Klebsiella pneumoniae; plasmid transfer; carbapenemases Carbapenem-resistant
Enterobacterales (CRE) is a critical public health problem in South America, where
the prevalence of NDM metallo-betalactamases has increased substantially in recent
years. In this study, we used whole genome sequencing to characterize a multidrug-
resistant (MDR) Klebsiella pneumoniae (UCO-361 strain) clinical isolate from a
teaching hospital in Chile. Using long-read (Nanopore) and short-read (Illumina)
sequence data, we identified a novel un-typeable megaplasmid (314,976 kb, pNDM-
1_UCO-361) carrying the blaNDM-1 carbapenem resistance gene within a Tn3000
transposon. Strikingly, conjugal transfer of pNDM-1_UCO-361 plasmid only occurs at
low temperatures with a high frequency of 4.3 × 10−6
transconjugants/receptors at 27 °C. UCO-361 belonged to the ST1588 clone,
previously identified in Latin America, and harbored aminoglycoside, extended-
spectrum β-lactamases (ESBLs), carbapenem, and quinolone-resistance
determinants. These findings suggest that blaNDM-1-bearing megaplasmids can be
adapted to carriage by some K. pneumoniae lineages, whereas its conjugation at low
temperatures could contribute to rapid dissemination at the
human–environmental interface. Laboratorio de Investigación en Agentes
Antibacterianos (LIAA-UdeC), Facultad de Ciencias Biológicas, Universidad de
Concepción, Concepción 4030000, Chile marioquezada@udec.cl; andopazo@udec.cl;
lincopan@usp.br; fernandaesposito@usp.br; bruna.fuga@hotmail.com;
pignatio@outlook.com; giseriedel@udec.cl; cfernandes@udec.cl; hbello@udec.cl;
marcelacifuentesdiaz@gmail.com; fsilva@hcuch.cl; borisbarrera68@gmail.com;
jchormazabal@ispch.cl; ggonzal@udec.cl 10.3390/antibiotics11091207 2022 11
9 - - 1207 -
Song, Zichen; Liang, Yuan; Yang, Jing Nanopore Detection Assisted DNA
Information Processing Nanomaterials EN Review nanopore detection; DNA
storage; ONT nanopores; artificial intelligence; DNA information processing The
deoxyribonucleotide (DNA) molecule is a stable carrier for large amounts of genetic
information and provides an ideal storage medium for next-generation information
processing technologies. Technologies that process DNA information, representing a
cross-disciplinary integration of biology and computer techniques, have become
attractive substitutes for technologies that process electronic information alone.
The detailed applications of DNA technologies can be divided into three components:
storage, computing, and self-assembly. The quality of DNA information processing
relies on the accuracy of DNA reading. Nanopore detection allows researchers to
accurately sequence nucleotides and is thus widely used to read DNA. In this paper,
we introduce the principles and development history of nanopore detection and
conduct a systematic review of recent developments and specific applications in DNA
information processing involving nanopore detection and nanopore-based storage. We
also discuss the potential of artificial intelligence in nanopore detection and DNA
information processing. This work not only provides new avenues for future nanopore
detection development, but also offers a foundation for the construction of more
advanced DNA information processing technologies. School of Control and
Computer Engineering, North China Electric Power University, Beijing 102206, China
zichensongug@163.com; 18811658276@163.com; yjzcdd_2000@ncepu.edu.cn
10.3390/nano12183135 2022 12 18 - - 3135 -
Geng, Yangyang; Zhang, Shixin; Yang, Ningxian; Qin, Likang Whole-Genome Sequencing
and Comparative Genomics Analysis of the Wild Edible Mushroom (Gomphus purpuraceus)
Provide Insights into Its Potential Food Application and Artificial Domestication
Genes EN Article Gomphus purpuraceus (Iwade) Yokoyama; edible fungi;
CAZymes; phylogenetic; secondary metabolisms Gomphus purpuraceus (Iwade)
Yokoyama is a species of wild fungi that grows in southwest China, considered an
edible and medicinal fungus with potential commercial prospects. However, the
detailed mechanisms related to the development of mycelium and the formation of the
fruiting body are unclear. To obtain a comprehensive overview of genetic features,
whole-genome and comparative genomics analyses of G. purpuraceus were performed.
High-quality DNA was extracted from the mycelium, which was isolated from a fresh
fruiting body of G. purpuraceus. The DNA sample was subjected to sequencing using
Illumina and Oxford Nanopore sequencing platforms. A genome assembly totaling 40.15
Mb in 50 contigs with an N50 length of 2.06 Mb was generated, and 8705 putative
predicted genes were found. Subsequently, phylogenetic analysis revealed a close
evolutionary relationship between G. purpuraceus and Gomphus bonarii. Moreover, a
total of 403 carbohydrate-active enzymes (CAZymes) were identified in G.
purpuraceus, which included 147 glycoside hydrolases (GHs), 85 glycosyl
transferases (GTs), 8 polysaccharide lyases (PLs), 76 carbohydrate esterases (CEs),
57 auxiliary activities (AAs) and 30 carbohydrate-binding modules (CBMs). Compared
with the other 13 fungi (Laccaria bicolor, Russula virescens, Boletus edulis,
etc.), the number and distribution of CAZymes in G. purpuraceus were similar to
other mycorrhizal fungi. Furthermore, the optimization of culture medium for G.
purpuraceus showed the efficient utilization of disaccharides such as sucrose and
maltose. The genome of G. purpuraceus provides new insights into its niche, food
applications and potential artificial domestication. Key Laboratory of Plant
Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of
Education), College of Life Sciences/Institute of Agro-Bioengineering, Guizhou
University, Hangtian West Road, Guiyang 550025, China yygengfood@sina.cn;
zsx262@126.com; nxyang2020@126.com; lkqin@gzu.edu.cn 10.3390/genes13091628 2022
13 9 - - 1628 -
Papa Mze, Nasserdine; Beye, Mamadou; Kacel, Idir; Tola, Raphael; Basco, Leonardo;
Bogreau, Hervé; Colson, Philippe; Fournier, Pierre-Edouard Simultaneous SARS-CoV-2
Genome Sequencing of 384 Samples on an Illumina MiSeq Instrument through Protocol
Optimization Genes EN Article Illumina MiSeq; SARS-CoV-2; next-
generation sequencing; genomics; sequence analysis In the present study, we
propose a high-throughput sequencing protocol using aNextera XT Library DNA kit on
an Illumina MiSeq instrument. We made major modifications to this library
preparation in order to multiplex 384 samples in a single Illumina flow cell. To
validate our protocol, we compared the sequences obtained with the modified
Illumina protocol to those obtained with the GridION Nanopore protocol. For the
modified Illumina protocol, our results showed that 94.9% (357/376) of the
sequences were interpretable, with a viral genome coverage between 50.5% and 99.9%
and an average depth of 421×. For the GridION Nanopore protocol, 94.6%
(356/376) of the sequences were interpretable, with a viral genome coverage between
7.0% and 98.6% and an average depth of 2123×. The modified Illumina protocol
allows for gaining EUR 4744 and returning results of 384 samples in 53.5 h versus
four times 55.5 h with the standard Illumina protocol. Our modified MiSeq protocol
yields similar genome sequence data as the GridION Nanopore protocol and has the
advantage of being able to handle four times more samples simultaneously and hence
is much less expensive. UMR VITROME, Aix-Marseille University, IRD, AP-HM, SSA, IHU
—Méditerranée Infection, 13005 Marseille, France npapamze@gmail.com;
bemamadou@gmail.com; idir.kacel@gmail.com; raphael.tola@ap-hm.fr; lkbasco@yahoo.fr;
hervebogreau@yahoo.fr; philippe.colson@univ-amu.fr; pierre-edouard.fournier@univ-
amu.fr 10.3390/genes13091648 2022 13 9 - - 1648 -
Hain, Carsten; Stadler, Rudolf; Kalinowski, Jörn Unraveling the Structural
Variations of Early-Stage Mycosis Fungoides—CD3 Based Purification and Third
Generation Sequencing as Novel Tools for the Genomic Landscape in CTCL Cancers
EN Article cutaneous T-cell lymphoma; mycosis fungoides; enrichment;
sequencing; nanopore; copy-number variation; structural variation Mycosis
fungoides (MF) is the most common cutaneous T-cell lymphoma (CTCL). At present,
knowledge of genetic changes in early-stage MF is insufficient. Additionally, low
tumor cell fraction renders calling of copy-number variations as the predominant
mutations in MF challenging, thereby impeding further investigations. We show that
enrichment of T cells from a biopsy of a stage I MF patient greatly increases tumor
fraction. This improvement enables accurate calling of recurrent MF copy-number
variants such as ARID1A and CDKN2A deletion and STAT5 amplification, undetected in
the unprocessed biopsy. Furthermore, we demonstrate that application of long-read
nanopore sequencing is especially useful for the structural variant rich CTCL. We
detect the structural variants underlying recurrent MF copy-number variants and
show phasing of multiple breakpoints into complex structural variant haplotypes.
Additionally, we record multiple occurrences of templated insertion structural
variants in this sample. Taken together, this study suggests a workflow to make the
early stages of MF accessible for genetic analysis, and indicates long-read
sequencing as a major tool for genetic analysis for MF. Center for Biotechnology
(CeBiTec), Bielefeld University, 33615 Bielefeld, Germany chain@cebitec.uni-
bielefeld.de; rudolf.stadler@ruhr-uni-bochum.de; joern@cebitec.uni-bielefeld.de
10.3390/cancers14184466 2022 14 18 - - 4466 -
Leiva, Ana; Chittarath, Khonesavanh; Lopez-Alvarez, Diana; Vongphachanh, Pinkham;
Gomez, Maria; Sengsay, Somkhit; Wang, Xiao-Wei; Rodriguez, Rafael; Newby, Jonathan;
Cuellar, Wilmer Mitochondrial Genetic Diversity of Bemisia tabaci (Gennadius)
(Hemiptera: Aleyrodidae) Associated with Cassava in Lao PDRInsects EN
Article Bemisia tabaci; whitefly; nanopore; mtCOI; Southeast Asia;
haplotype; Cassava Mosaic Disease Cassava Mosaic Disease (CMD) caused by Sri
Lankan cassava mosaic virus (SLCMV), has rapidly spread in Southeast Asia (SEA)
since 2016. Recently it has been documented in Lao PDR. Previous reports have
identified whitefly species of B. tabaci as potential vectors of CMD in SEA, but
their occurrence and distribution in cassava fields is not well known. We conducted
a countrywide survey in Lao PDR for adult whiteflies in cassava fields, and
determined the abundance and genetic diversity of the B. tabaci species complex
using mitochondrial cytochrome oxidase I (mtCOI) sequencing. In order to expedite
the process, PCR amplifications were performed directly on whitefly adults without
DNA extraction, and mtCOI sequences obtained using nanopore portable-sequencing
technology. Low whitefly abundances and two cryptic species of the B. tabaci
complex, Asia II 1 and Asia II 6, were identified. This is the first work on
abundance and genetic identification of whiteflies associated with cassava in Lao
PDR. This study indicates currently only a secondary role for Asia II in spreading
CMD or as a pest. Routine monitoring and transmission studies on Asia II 6 should
be carried out to establish its potential role as a vector of SLCMV in this region.
Cassava Program, Crops for Nutrition and Health, International Center for
Tropical Agriculture (CIAT), The Americas Hub, Km 17 Recta Cali-Palmira, Cali
763537, Colombia a.m.leiva@cgiar.org; chittarhat_2005@yahoo.com;
dilopezal@unal.edu.co; pinkhamvpc@gmail.com; m.i.gomez@cgiar.org;
somkhitsengsay@hotmail.com; xwwang@zju.edu.cn; rafael.rodriguez@cgiar.org;
j.newby@cgiar.org; w.cuellar@cgiar.org 10.3390/insects13100861 2022 13 10
- - 861 -
Iossi, Matheus; Palú, Isabela; Soares, Douglas; Vieira, Wagner; Alves, Lucas;
Stevani, Cassius; Caitano, Cinthia; Atum, Samir; Freire, Renato; Dias, Eustáquio;
Zied, Diego Metaprofiling of the Bacterial Community in Colonized Compost Extracts
by Agaricus subrufescens Journal of Fungi EN Article Agaricus blazei;
metagenomics; microbiomics; mushroom production; 16S rDNA; nanopore sequencing
It is well-known that bacteria and fungi play important roles in the
relationships between mycelium growth and the formation of fruiting bodies. The sun
mushroom, Agaricus subrufescens, was discovered in Brazil ca. 1960 and it has
become known worldwide due to its medicinal and nutritional properties. This work
evaluated the bacterial community present in mushroom-colonized compost extract
(MCCE) prepared from cultivation of A. subrufescens, its dynamics with two
different soaking times and the influence of the application of those extracts on
the casing layer of a new compost block for A. subrufescens cultivation. MCCEs were
prepared through initial submersion of the colonized compost for 1 h or 24 h in
water followed by application on casing under semi-controlled conditions. Full-
length 16S rRNA genes of 1 h and 24 h soaked MCCE were amplified and sequenced
using nanopore technology. Proteobacteria, followed by Firmicutes and
Planctomycetes, were found to be the most abundant phyla in both the 1 h and 24 h
soaked MCCE. A total of 275 different bacterial species were classified from 1 h
soaked MCCE samples and 166 species from 24 h soaked MCCE, indicating a decrease in
the bacterial diversity with longer soaking time during the preparation of MCCE.
The application of 24 h soaked MCCE provided increases of 25% in biological
efficiency, 16% in precociousness, 53% in the number of mushrooms and 40% in
mushroom weight compared to control. Further investigation is required to determine
strategies to enhance the yield and quality of the agronomic traits in commercial
mushroom cultivation. Programa de Pós-Graduação em Microbiologia Agropecuária,
Faculdade de Ciências Agrárias e Veterinárias (FCAV), Universidade Estadual
Paulista (UNESP), São Paulo 14884-900, Brazil matheusiossi1@gmail.com;
isabela.a.palu@gmail.com; douglas@iq.usp.br; wagnergvj@gmail.com;
lucasagro@live.com; stevani@iq.usp.br; cinthia.ecardoso@hotmail.com;
samir.atum@usp.br; rsfreire@iq.usp.br; esdias@ufla.br; dczied@gmail.com
10.3390/jof8100995 2022 8 10 - - 995 -
Monecke, Stefan; Roberts, Marilyn; Braun, Sascha; Diezel, Celia; Müller, Elke;
Reinicke, Martin; Linde, Jörg; Joshi, Prabhu; Paudel, Saroj; Acharya, Mahesh;
Chalise, Mukesh; Feßler, Andrea; Hotzel, Helmut; Khanal, Laxman; Koju, Narayan;
Schwarz, Stefan; Kyes, Randall; Ehricht, Ralf Sequence Analysis of Novel
Staphylococcus aureus Lineages from Wild and Captive Macaques International
Journal of Molecular Sciences EN Article Staphylococcus aureus; Macaca spp.;
macaques; next-generation sequencing Staphylococcus aureus is a widespread and
common opportunistic bacterium that can colonise or infect humans as well as a wide
range of animals. There are a few studies of both methicillin-susceptible S. aureus
(MSSA) and methicillin-resistant S. aureus (MRSA) isolated from monkeys, apes, and
lemurs, indicating a presence of a number of poorly or unknown lineages of the
pathogen. In order to obtain insight into staphylococcal diversity, we sequenced
strains from wild and captive individuals of three macaque species (Macaca mulatta,
M. assamensis, and M. sylvanus) using Nanopore and Illumina technologies. These
strains were previously identified by microarray as poorly or unknown strains.
Isolates of novel lineages ST4168, ST7687, ST7688, ST7689, ST7690, ST7691, ST7692,
ST7693, ST7694, ST7695, ST7745, ST7746, ST7747, ST7748, ST7749, ST7750, ST7751,
ST7752, ST7753, and ST7754 were sequenced and characterised for the first time. In
addition, isolates belonging to ST2990, a lineage also observed in humans, and
ST3268, a MRSA strain already known from macaques, were also included into the
study. Mobile genetic elements, genomic islands, and carriage of prophages were
analysed. There was no evidence for novel host-specific virulence factors. However,
a conspicuously high rate of carriage of a pathogenicity island harbouring edinB
and etD2/etE as well as a higher number of repeat units within the gene sasG
(encoding an adhesion factor) than in human isolates were observed. None of the
strains harboured the genes encoding Panton–Valentine leukocidin. In
conclusion, wildlife including macaques may harbour an unappreciated diversity of
S. aureus lineages that may be of clinical relevance for humans, livestock, or for
wildlife conservation, given the declining state of many wildlife populations.
Leibniz Institute of Photonic Technology (IPHT), 07745 Jena, Germany
stefan.monecke@leibniz-ipht.de; marilynr@uw.edu; sascha.braun@leibniz-
ipht.de; celia.diezel@leibniz-ipht.de; elke.mueller@leibniz-ipht.de;
martin.reinicke@leibniz-ipht.de; joerg.linde@fli.de; cmilanjoshi@gmail.com;
pulu.saroj@gmail.com; maheshacharya045@gmail.com; mukesh57@hotmail.com;
andrea.fessler@fu-berlin.de; h-hotzel@t-online.de; lkhanal@cdztu.edu.np;
npkoju.2003@gmail.com; stefan.schwarz@fu-berlin.de; rkyes@uw.edu;
ralf.ehricht@leibniz-ipht.de 10.3390/ijms231911225 2022 23 19 - -
11225 -
Waite, David; Liefting, Lia; Delmiglio, Catia; Chernyavtseva, Anastasia; Ha, Hye;
Thompson, Jeremy Development and Validation of a Bioinformatic Workflow for the
Rapid Detection of Viruses in Biosecurity Viruses EN Article biosecurity;
virology; bioinformatics; high-throughput sequencing; Oxford Nanopore; Illumina;
DNA; RNA The field of biosecurity has greatly benefited from the widespread
adoption of high-throughput sequencing technologies, for its ability to deeply
query plant and animal samples for pathogens for which no tests exist. However, the
bioinformatics analysis tools designed for rapid analysis of these sequencing
datasets are not developed with this application in mind, limiting the ability of
diagnosticians to standardise their workflows using published tool kits. We sought
to assess previously published bioinformatic tools for their ability to identify
plant- and animal-infecting viruses while distinguishing from the host genetic
material. We discovered that many of the current generation of virus-detection
pipelines are not adequate for this task, being outperformed by more generic
classification tools. We created synthetic MinION and HiSeq libraries simulating
plant and animal infections of economically important viruses and assessed a series
of tools for their suitability for rapid and accurate detection of infection, and
further tested the top performing tools against the VIROMOCK Challenge dataset to
ensure that our findings were reproducible when compared with international
standards. Our work demonstrated that several methods provide sensitive and
specific detection of agriculturally important viruses in a timely manner and
provides a key piece of ground truthing for method development in this space.
Plant Health and Environment Laboratory, Ministry for Primary Industries,
P.O. Box 2095, Auckland 1140, New Zealand david.waite@mpi.govt.nz;
lia.liefting@mpi.govt.nz; catia.delmiglio@mpi.govt.nz;
anastasia.chernyavtseva@mpi.govt.nz; hyejeong.ha@mpi.govt.nz;
jeremy.thompson@mpi.govt.nz 10.3390/v14102163 2022 14 10 - - 2163
 -
Player, Robert; Verratti, Kathleen; Staab, Andrea; Forsyth, Ellen; Ernlund, Amanda;
Joshi, Mihir; Dunning, Rebecca; Rozak, David; Grady, Sarah; Goodwin, Bruce;
Sozhamannan, Shanmuga Optimization of Oxford Nanopore Technology Sequencing
Workflow for Detection of Amplicons in Real Time Using ONT-DART Tool Genes EN
Communication biodefense; biodetection; biosurveillance; biothreat
agents; oxford nanopore sequencing; real-time Sequencing; sequencing library
preparation An optimized, well-tested and validated targeted genomic sequencing-
based high-throughput assay is currently not available ready for routine biodefense
and biosurveillance applications. Earlier, we addressed this gap by developing and
establishing baseline comparisons of a multiplex end-point Polymerase Chain
Reaction (PCR) assay followed by Oxford Nanopore Technology (ONT) based amplicon
sequencing to real time PCR and customized data processing. Here, we expand upon
this effort by identifying the optimal ONT library preparation method for
integration into a novel software platform ONT-DART (ONT-Detection of Amplicons in
Real-Time). ONT-DART is a dockerized, real-time, amplicon-sequence analysis
workflow that is used to reproducibly process and filter read data to support
actionable amplicon detection calls based on alignment metrics, within sample
statistics, and no-template control data. This analysis pipeline was used to
compare four ONT library preparation protocols using R9 and Flongle (FL) flow
cells. The two 4-Primer methods tested required the shortest preparation times (5.5
and 6.5 h) for 48 libraries but provided lower fidelity data. The Native Barcoding
and Ligation methods required longer preparation times of 8 and 12 h, respectively,
and resulted in higher overall data quality. On average, data derived from R9 flow
cells produced true positive calls for target organisms more than twice as fast as
the lower throughput FL flow cells. These results suggest that utilizing the R9
flowcell with an ONT Native Barcoding amplicon library method in combination with
ONT-DART platform analytics provides the best sequencing-based alternative to
current PCR-based biodetection methods. Applied Physics Laboratory, The Johns
Hopkins University, Laurel, MD 20723, USA robert.player@datirium.com;
kathleen.verratti@jhuapl.edu; andrea.b.staab.civ@us.navy.mil;
ellen.forsyth@jhuapl.edu; amanda.ernlund@jhuapl.edu; mihir.joshi@jhuapl.edu;
becky.a.dunning@gsk.com; david.a.rozak2.civ@health.mil; sarah.grady@jhuapl.edu;
bruce.g.goodwin4.civ@army.mil; shanmuga.sozhamannan.ctr@army.mil
10.3390/genes13101785 2022 13 10 - - 1785 -
Maboni, Grazieli; Baptista, Rodrigo; Wireman, Joy; Framst, Isaac; Summers, Anne;
Sanchez, Susan Three Distinct Annotation Platforms Differ in Detection of
Antimicrobial Resistance Genes in Long-Read, Short-Read, and Hybrid Sequences
Derived from Total Genomic DNA or from Purified Plasmid DNAAntibiotics EN
Article AMR prediction; plasmids; Nanopore sequencing; Illumina
sequencing; whole genomes; WGS workflows Recent advances and lower costs in rapid
high-throughput sequencing have engendered hope that whole genome sequencing (WGS)
might afford complete resistome characterization in bacterial isolates. WGS is
particularly useful for the clinical characterization of fastidious and slow-
growing bacteria. Despite its potential, several challenges should be addressed
before adopting WGS to detect antimicrobial resistance (AMR) genes in the clinical
laboratory. Here, with three distinct ESKAPE bacteria (Enterococcus faecium,
Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas
aeruginosa, and Enterobacter spp.), different approaches were compared to identify
best practices for detecting AMR genes, including: total genomic DNA and plasmid
DNA extractions, the solo assembly of Illumina short-reads and of Oxford Nanopore
Technologies (ONT) long-reads, two hybrid assembly pipelines, and three in silico
AMR databases. We also determined the susceptibility of each strain to 21
antimicrobials. We found that all AMR genes detected in pure plasmid DNA were also
detectable in total genomic DNA, indicating that, at least in these three
enterobacterial genera, the purification of plasmid DNA was not necessary to detect
plasmid-borne AMR genes. Illumina short-reads used with ONT long-reads in either
hybrid or polished assemblies of total genomic DNA enhanced the sensitivity and
accuracy of AMR gene detection. Phenotypic susceptibility closely corresponded with
genotypes identified by sequencing; however, the three AMR databases differed
significantly in distinguishing mobile dedicated AMR genes from non-mobile
chromosomal housekeeping genes in which rare spontaneous resistance mutations might
occur. This study indicates that each method employed in a WGS workflow has an
impact on the detection of AMR genes. A combination of short- and long-reads,
followed by at least three different AMR databases, should be used for the
consistent detection of such genes. Further, an additional step for plasmid DNA
purification and sequencing may not be necessary. This study reveals the need for
standardized biochemical and informatic procedures and database resources for
consistent, reliable AMR genotyping to take full advantage of WGS in order to
expedite patient treatment and track AMR genes within the hospital and community.
Athens Veterinary Diagnostic Laboratory, University of Georgia, Athens, GA
30602, USA grazieli.maboni@gmail.com; rodrigopbaptista@gmail.com; wireman@uga.edu;
iframst@uoguelph.ca; summers@uga.edu; ssanchez@uga.edu
10.3390/antibiotics11101400 2022 11 10 - - 1400 -
Liu, Dong; Gui, Lang; Zhu, Yefei; Xu, Cong; Zhou, Wenzong; Li, Mingyou Chromosome-
Level Assembly of Male Opsariichthys bidens Genome Provides Insights into the
Regulation of the GnRH Signaling Pathway and Genome Evolution Biology EN
Article Cyprinid fish; hook snout carp; sexual dimorphism; comparative
genomics; GnRH signaling The hook snout carp Opsariichthys bidens is an
important farmed fish in East Asia that shows sexual dimorphism in growth, with
males growing faster and larger than females. To understand these complex traits
and improve molecular breeding, chromosome-level genome assembly of male O. bidens
was performed using Illumina, Nanopore, and Hi-C sequencing. The 992.9 Mb genome
sequences with a contig N50 of 5.2 Mb were anchored to 38 chromosomes corresponding
to male karyotypes. Of 30,922 functionally annotated genes, 97.5% of BUSCO genes
were completely detected. Genome evolution analysis showed that the expanded and
contracted gene families in the male O. bidens genome were enriched in 76 KEGG
pathways, and 78 expanded genes were involved in the GnRH signaling pathway that
regulates the synthesis and secretion of luteinizing hormone and glycoprotein
hormones, further acting on male growth by inducing growth hormone. Compared to the
released female O. bidens genome, the number of annotated genes in males was much
higher (23,992). The male chromosome LG06 exhibited over 97% identity with the
female GH14/GH38. Male-specific genes were identified for LG06, where structural
variation, including deletions and insertions, occurred at a lower rate, suggesting
a centric fusion of acrocentric chromosomes GH14 and GH38. The genome-synteny
analysis uncovered significant inter-chromosome conservation between male O. bidens
and grass carp, the former originating from ancestral chromosome breakage to
increase the chromosome number. Our results provide a valuable genetic resource for
studying the regulation of sexual dimorphism, sex-determining mechanisms, and
molecular-guided breeding of O. bidens. Key Laboratory of Integrated Rice-Fish
Farming, Ministry of Agriculture and Rural Affairs, Shanghai Ocean University,
Shanghai 201306, China dliu@shou.edu.cn; lgui@shou.edu.cn; yesfigo@163.com;
m200100050@st.shou.edu.cn; zhouwz001@163.com; myli@shou.edu.cn
10.3390/biology11101500 2022 11 10 - - 1500 -
Sedaghat-Hamedani, Farbod; Rebs, Sabine; Kayvanpour, Elham; Zhu, Chenchen; Amr,
Ali; Müller, Marion; Haas, Jan; Wu, Jingyan; Steinmetz, Lars; Ehlermann, Philipp;
Streckfuss-Bömeke, Katrin; Frey, Norbert; Meder, Benjamin Genotype Complements the
Phenotype: Identification of the Pathogenicity of an LMNA Splice Variant by
Nanopore Long-Read Sequencing in a Large DCM Family International Journal of
Molecular Sciences EN Article familial DCM; laminopathy; long-read
sequencing; nanopore; induced pluripotent stem cell cardiomyocytes Dilated
cardiomyopathy (DCM) is a common cause of heart failure (HF) and is of familial
origin in 20–40% of cases. Genetic testing by next-generation sequencing
(NGS) has yielded a definite diagnosis in many cases; however, some remain elusive.
In this study, we used a combination of NGS, human-induced pluripotent-stem-cell-
derived cardiomyocytes (iPSC-CMs) and nanopore long-read sequencing to identify the
causal variant in a multi-generational pedigree of DCM. A four-generation family
with familial DCM was investigated. Next-generation sequencing (NGS) was performed
on 22 family members. Skin biopsies from two affected family members were used to
generate iPSCs, which were then differentiated into iPSC-CMs. Short-read RNA
sequencing was used for the evaluation of the target gene expression, and long-read
RNA nanopore sequencing was used to evaluate the relevance of the splice variants.
The pedigree suggested a highly penetrant, autosomal dominant mode of inheritance.
The phenotype of the family was suggestive of laminopathy, but previous genetic
testing using both Sanger and panel sequencing only yielded conflicting evidence
for LMNA p.R644C (rs142000963), which was not fully segregated. By re-sequencing
four additional affected family members, further non-coding LMNA variants could be
detected: rs149339264, rs199686967, rs201379016, and rs794728589. To explore the
roles of these variants, iPSC-CMs were generated. RNA sequencing showed the LMNA
expression levels to be significantly lower in the iPSC-CMs of the LMNA variant
carriers. We demonstrated a dysregulated sarcomeric structure and altered calcium
homeostasis in the iPSC-CMs of the LMNA variant carriers. Using targeted nanopore
long-read sequencing, we revealed the biological significance of the variant
c.356+1G>A, which generates a novel 5′ splice site in exon 1 of the
cardiac isomer of LMNA, causing a nonsense mRNA product with almost complete RNA
decay and haploinsufficiency. Using novel molecular analysis and nanopore
technology, we demonstrated the pathogenesis of the rs794728589 (c.356+1G>A)
splice variant in LMNA. This study highlights the importance of precise diagnostics
in the clinical management and workup of cardiomyopathies. Institute for
Cardiomyopathies Heidelberg (ICH), University Hospital Heidelberg, 69120
Heidelberg, Germany farbod.sedaghat-hamedani@med.uni-heidelberg.de;
sabine.rebs@med.uni-goettingen.de; elham.kayvanpour@med.uni-heidelberg.de;
czhu5@stanford.edu; ali.amr@med.uni-heidelberg.de; mamueller@hdz-nrw.de;
jan.haas@med.uni-heidelberg.de; jingyanwu1987@gmail.com;
lars.steinmetz@stanford.edu; philipp.ehlermann@med.uni-heidelberg.de;
katrin.streckfuss@med.uni-goettingen.de; norbert.frey@med.uni-heidelberg.de;
benjamin.meder@med.uni-heidelberg.de 10.3390/ijms232012230 2022 23 20
- - 12230 -
Mackie, Joanne; Kinoti, Wycliff; Chahal, Sumit; Lovelock, David; Campbell, Paul;
Tran-Nguyen, Lucy; Rodoni, Brendan; Constable, Fiona Targeted Whole Genome
Sequencing (TWG-Seq) of Cucumber Green Mottle Mosaic Virus Using Tiled Amplicon
Multiplex PCR and Nanopore Sequencing Plants EN Article CGMMV; tiled
amplicon sequencing; nanopore Rapid and reliable detection tools are essential for
disease surveillance and outbreak management, and genomic data is essential to
determining pathogen origin and monitoring of transmission pathways. Low virus copy
number and poor RNA quality can present challenges for genomic sequencing of plant
viruses, but this can be overcome by enrichment of target nucleic acid. A targeted
whole genome sequencing (TWG-Seq) approach for the detection of cucumber green
mottle mosaic virus (CGMMV) has been developed where overlapping amplicons
generated using two multiplex RT-PCR assays are then sequenced using the Oxford
Nanopore MinION. Near complete coding region sequences were assembled with
≥100× coverage for infected leaf tissue dilution samples with RT-qPCR
cycle quantification (Cq) values from 11.8 to 38 and in seed dilution samples with
Cq values 13.8 to 27. Consensus sequences assembled using this approach showed
greater than 99% nucleotide similarity when compared to genomes produced using
metagenomic sequencing. CGMMV could be confidently detected in historical seed
isolates with degraded RNA. Whilst limited access to, and costs associated with
second-generation sequencing platforms can influence diagnostic outputs, the
portable Nanopore technology offers an affordable high throughput sequencing
alternative when combined with TWG-Seq for low copy or degraded samples. School
of Applied Systems Biology, La Trobe University, Melbourne, VIC 3083, Australia
joanne.mackie@ecodev.vic.gov.au; cliff.kinoti@agriculture.vic.gov.au;
sumit.chahal@ecodev.vic.gov.au; david.lovelock@agriculture.vic.gov.au;
paul.campbell@daf.qld.gov.au; ltran-nguyen@phau.com.au;
brendan.rodoni@agriculture.vic.gov.au; fiona.constable@agriculture.vic.gov.au
10.3390/plants11202716 2022 11 20 - - 2716 -
Xu, Fu; Li, Xiuxiu; Ren, Hui; Zeng, Rensen; Wang, Zhoutao; Hu, Hongli; Bao,
Jiandong; Que, Youxiong The First Telomere-to-Telomere Chromosome-Level Genome
Assembly of Stagonospora tainanensis Causing Sugarcane Leaf Blight Journal of
Fungi EN Article Stagonospora tainanensis; sugarcane leaf blight;
pathogenicity; Nanopore sequencing; genome assembly The sexual morph
Leptosphaeria taiwanensis Yen and Chi and its asexual morph Stagonospora
tainanensis W. H. Hsieh is an important necrotrophic fungal phytopathogen, which
causes sugarcane leaf blight, resulting in loss of cane tonnage and sucrose in
susceptible sugarcane varieties. Decoding the genome and understanding of the basis
of virulence is vitally important for devising effective disease control
strategies. Here, we present a 38.25-Mb high-quality genome assembly of S.
tainanensis strain StFZ01, denovo assembled with 10.19 Gb Nanopore sequencing long
reads (~267×) and 3.82 Gb Illumina short reads (~100×). The genome
assembly consists of 12 contigs with N50 of 2.86 Mb of which 5 belong to the
telomere to telomere (T2T) chromosome. It contains 13.20% repeat sequences, 12,543
proteins, and 12,206 protein-coding genes with the BUSCO completeness 99.18% at
fungi (n = 758) and 99.87% at ascomycota (n = 1706), indicating the high accuracy
and completeness of our gene annotations. The virulence analysis in silico revealed
the presence of 2379 PHIs, 599 CAZys, 248 membrane transport proteins, 191
cytochrome P450 enzymes, 609 putative secreted proteins, and 333 effectors in the
StFZ01 genome. The genomic resources presented here will not only be helpful for
development of specific molecular marker and diagnosis technique, population
genetics, molecular taxonomy, and disease managements, it can also provide a
significant precise genomic reference for investigating the ascomycetous genome,
the necrotrophic lifestyle, and pathogenicity in the future. Key Lab of
Sugarcane Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs,
Fuzhou 350002, China xufu1219@126.com; lixiuxiu891012@163.com;
hui224364@163.com; rszeng@fafu.edu.cn; wzt1417@126.com; huhongli7905@gmail.com;
baojd@zaas.ac.cn; queyouxiong@126.com 10.3390/jof8101088 2022 8 10
- - 1088 -
Pot, Matthieu; Reynaud, Yann; Couvin, David; Dereeper, Alexis; Ferdinand, Séverine;
Bastian, Sylvaine; Foucan, Tania; Pommier, Jean-David; Valette, Marc; Talarmin,
Antoine; Guyomard-Rabenirina, Stéphanie; Breurec, SébastienEmergence of a Novel
Lineage and Wide Spread of a blaCTX-M-15/IncHI2/ST1 Plasmid among Nosocomial
Enterobacter in Guadeloupe Antibiotics EN Article Caribbean; Enterobacter
cloacae complex; ESBL; healthcare; hsp60; molecular sequencing; Nanopore; plasmid;
ST114; ST1503 Between April 2018 and August 2019, a total of 135 strains of
Enterobacter cloacae complex (ECC) were randomly collected at the University
Hospital Center of Guadeloupe to investigate the structure and diversity of the
local bacterial population. These nosocomial isolates were initially identified
genetically by the hsp60 typing method, which revealed the clinical relevance of E.
xiangfangensis (n = 69). Overall, 57/94 of the third cephalosporin-resistant
strains were characterized as extended-spectrum-β-lactamase (ESBL) producers,
and their whole-genome was sequenced using Illumina technology to determine the
clonal relatedness and diffusion of resistance genes. We found limited genetic
diversity among sequence types (STs). ST114 (n = 13), ST1503 (n = 9), ST53 (n = 5)
and ST113 (n = 4), which belong to three different Enterobacter species, were the
most prevalent among the 57 ESBL producers. The blaCTXM-15 gene was the most
prevalent ESBL determinant (56/57) and was in most cases associated with IncHI2/ST1
plasmid replicon carriage (36/57). To fully characterize this predominant blaCTXM-
15/IncHI2/ST1 plasmid, four isolates from different lineages were also sequenced
using Oxford Nanopore sequencing technology to generate long-reads. Hybrid sequence
analyses confirmed the circulation of a well-conserved plasmid among ECC members.
In addition, the novel ST1503 and its associated species (ECC taxon 4) were
analyzed, in view of its high prevalence in nosocomial infections. These genetic
observations confirmed the overall incidence of nosocomial ESBL Enterobacteriaceae
infections acquired in this hospital during the study period, which was clearly
higher in Guadeloupe (1.59/1000 hospitalization days) than in mainland France
(0.52/1,000 hospitalization days). This project revealed issues and future
challenges for the management and surveillance of nosocomial and multidrug-
resistant Enterobacter in the Caribbean. Transmission, Reservoir and Diversity of
Pathogens Unit, Pasteur Institute of Guadeloupe, 97139 Les Abymes, France
matthieu.pot@ird.fr; yann.reynaud88@gmail.com; dcouvin@pasteur-guadeloupe.fr;
alexis.dereeper@ird.fr; sferdinand@pasteur-guadeloupe.fr;
sylvainebastian@gmail.com; tania.foucan@chu-guadeloupe.fr; jdpommier@yahoo.fr;
marc.valette@chu-guadeloupe.fr; atalarmin@pasteur-guadeloupe.fr; sguyomard@pasteur-
guadeloupe.fr; sbreurec@gmail.com 10.3390/antibiotics11101443 2022 11 10
- - 1443 -
Ecovoiu, Alexandru; Bologa, Alexandru; Chifiriuc, David; Ciuca, Andrei; Constantin,
Nicoleta; Ghionoiu, Iulian; Ghita, Iulian; Ratiu, Attila Genome
ARTIST_v2—An Autonomous Bioinformatics Tool for Annotation of Natural
Transposons in Sequenced Genomes International Journal of Molecular Sciences
EN Article Drosophila melanogaster; Genome ARTIST; natural
transposons; insertion mapping; genome sequencing; bioinformatics The
annotation of transposable elements (transposons) is a very dynamic field of
genomics and various tools assigned to support this bioinformatics endeavor have
been developed and described. Genome ARTIST v1.19 (GA_v1.19) software was conceived
for mapping artificial transposons mobilized during insertional mutagenesis
projects, but the new functions of GA_v2 qualify it as a tool for the mapping and
annotation of natural transposons (NTs) in long reads, contigs and assembled
genomes. The tabular export of mapping and annotation data for high-throughput data
analysis, the generation of a list of flanking sequences around the coordinates of
insertion or around the target site duplications and the computing of a consensus
sequence for the flanking sequences are all key assets of GA_v2. Additionally, we
developed a set of scripts that enable the user to annotate NTs, to harness
annotations offered by FlyBase for Drosophila melanogaster genome, to convert
sequence files from .fasta to .raw, and to extract junction query sequences
essential for NTs mapping. Herein, we present the applicability of GA_v2 for a
preliminary annotation of P-element and hobo class II NTs and copia retrotransposon
in the genome of D. melanogaster strain Horezu_LaPeri (Horezu), Romania, which was
sequenced with Nanopore technology in our laboratory. We used contigs assembled
with Flye tool and a Q10 quality filter of the reads. Our results suggest that
GA_v2 is a reliable autonomous tool able to perform mapping and annotation of NTs
in genomes sequenced by long sequencing technology. GA_v2 is open-source software
compatible with Linux, Mac OS and Windows and is available at GitHub repository and
dedicated website. Department of Genetics, Faculty of Biology, University of
Bucharest, 060101 Bucharest, Romania alexandru.ecovoiu@bio.unibuc.ro;
alexandru.bologa@drd.unibuc.ro; david.chifiriuc@gmail.com; andrei.ciuca@gmail.com;
constantin.nicoleta-denisa@s.bio.unibuc.ro; iulian.ghionoiu@gmail.com;
ic.ghita@gmail.com; attila.ratiu@bio.unibuc.ro 10.3390/ijms232012686 2022 23
20 - - 12686 -
Buytaers, Florence; Verhaegen, Bavo; Gand, Mathieu; D’aes, Jolien; Vanneste, Kevin;
Roosens, Nancy; Marchal, Kathleen; Denayer, Sarah; De Keersmaecker, Sigrid
Metagenomics to Detect and Characterize Viruses in Food Samples at Genome
Level? Lessons Learnt from a Norovirus Study Foods EN Article
metagenomics; norovirus; food; typing; Oxford Nanopore sequencing; adaptive
sampling In this proof-of-concept study on food contaminated with norovirus, we
investigated the feasibility of metagenomics as a new method to obtain the whole
genome sequence of the virus and perform strain level characterization but also
relate to human cases in order to resolve foodborne outbreaks. We tested several
preparation methods to determine if a more open sequencing approach, i.e., shotgun
metagenomics, or a more targeted approach, including hybrid capture, was the most
appropriate. The genetic material was sequenced using Oxford Nanopore technologies
with or without adaptive sampling, and the data were analyzed with an in-house
bioinformatics workflow. We showed that a viral genome sequence could be obtained
for phylogenetic analysis with shotgun metagenomics if the contamination load was
sufficiently high or after hybrid capture for lower contamination. Relatedness to
human cases goes well beyond the results obtained with the current qPCR methods.
This workflow was also tested on a publicly available dataset of food spiked with
norovirus and hepatitis A virus. This allowed us to prove that we could detect even
fewer genome copies and two viruses present in a sample using shotgun metagenomics.
We share the lessons learnt on the satisfactory and unsatisfactory results in an
attempt to advance the field. Transversal Activities in Applied Genomics,
Sciensano, 1050 Brussels, Belgium florence.buytaers@sciensano.be;
bavo.verhaegen@sciensano.be; mathieu.gand@sciensano.be; jolien.daes@sciensano.be;
kevin.vanneste@sciensano.be; nancy.roosens@sciensano.be; kathleen.marchal@ugent.be;
sarah.denayer@sciensano.be; sigrid.dekeersmaecker@sciensano.be
10.3390/foods11213348 2022 11 21 - - 3348 -
Dvorianinova, Ekaterina; Bolsheva, Nadezhda; Pushkova, Elena; Rozhmina, Tatiana;
Zhuchenko, Alexander; Novakovskiy, Roman; Povkhova, Liubov; Sigova, Elizaveta;
Zhernova, Daiana; Borkhert, Elena; Kaluzhny, Dmitry; Melnikova, Nataliya; Dmitriev,
Alexey Isolating Linum usitatissimum L. Nuclear DNA Enabled Assembling High-
Quality Genome International Journal of Molecular Sciences EN Article
flax; Linum usitatissimum; nuclei extraction; high-molecular-weight DNA;
nanopore; high-quality genome High-quality genome sequences help to elucidate the
genetic basis of numerous biological processes and track species evolution. For
flax (Linum usitatissimum L.)—a multifunctional crop, high-quality assemblies
from Oxford Nanopore Technologies (ONT) data were unavailable, largely due to the
difficulty of isolating pure high-molecular-weight DNA. This article proposes a
scheme for gaining a contiguous L. usitatissimum assembly using Nanopore data. We
developed a protocol for flax nuclei isolation with subsequent DNA extraction,
which allows obtaining about 5 μg of pure high-molecular-weight DNA from 0.5 g
of leaves. Such an amount of material can be collected even from a single plant and
yields more than 30 Gb of ONT data in two MinION runs. We performed a comparative
analysis of different genome assemblers and polishers on the gained data and
obtained the final 447.1-Mb assembly of L. usitatissimum line 3896 genome using the
Canu—Racon (two iterations)—Medaka combination. The genome comprised
1695 contigs and had an N50 of 6.2 Mb and a completeness of 93.8% of BUSCOs from
eudicots_odb10. Our study highlights the impact of the chosen genome construction
strategy on the resulting assembly parameters and its eligibility for future
genomic studies. Engelhardt Institute of Molecular Biology, Russian Academy of
Sciences, Moscow 119991, Russia dvorianinova.em@phystech.edu;
nlbolsheva@mail.ru; pushkova18@gmail.com; tatyana_rozhmina@mail.ru;
ecovilar@mail.ru; 0legovich46@mail.ru; povhova.lv@phystech.edu;
sigova.ea@phystech.edu; zhernova.d@yandex.ru; sashai@inbox.ru; uzhny@mail.ru; mnv-
4529264@yandex.ru; alex_245@mail.ru 10.3390/ijms232113244 2022 23 21 -
- 13244 -
Dantas, Anna; Oliveira, Hellen; Gomes, Camila; Alves, Daniele; Infante, Priscilla;
Caitité, Rosimara; Fritsch, Hegger; Cucco, Marina; Silva, Lucas; Oliveira, Caline;
Bittencourt, Rafaela; Amorim, Aline; Nascimento, Ana; Marinho, Francely; de
Medeiros, Danielle; de Oliveira, Márcio; Mistro, Sostenes; de Melo, Fabricio;
Pereira, Taiana; Guimarães, Ana; Timenetsky, Jorge; Moreira, Pablo; de Oliveira,
Sandra; Alcantara, Luiz; Giovanetti, Marta; Santos, Luciane; Fonseca, Vagner;
Barreto, Fernanda; Campos, Guilherme; Marques, Lucas Retrospective Analysis of the
SARS-CoV-2 Infection Profile in COVID-19 Positive Patients in Vitoria da Conquista,
Northeast Brazil Viruses EN Article SARS-CoV-2; gene expressions;
sequencing; genomic and epidemiological surveillance Severe Acute Respiratory
Syndrome Coronavirus-2 (SARS-CoV-2) is responsible for causing Coronavirus Disease-
2019 (COVID-19), a heterogeneous clinical condition that manifests varying symptom
severity according to the demographic profile of the studied population. While many
studies have focused on the spread of COVID-19 in large urban centers in Brazil,
few have evaluated medium or small cities in the Northeast region. The aims of this
study were: (i) to identify risk factors for mortality from SARS-CoV-2 infection,
(ii) to evaluate the gene expression patterns of key immune response pathways using
nasopharyngeal swabs of COVID-19 patients, and (iii) to identify the circulating
SARS-CoV-2 variants in the residents of a medium-sized city in Northeast Brazil. A
total of 783 patients infected with SARS-CoV-2 between May 2020 and August 2021
were included in this study. Clinical-epidemiological data from patients who died
and those who survived were compared. Patients were also retrospectively divided
into three groups based on disease severity: asymptomatic, mild, and
moderate/severe. Samples were added to a qPCR array for analyses of 84 genes
involved with immune response pathways and sequenced using the Oxford Nanopore
MinION technology. Having pre-existing comorbidity; being male; having
cardiovascular disease, diabetes, and/or chronic obstructive pulmonary disease; and
PCR cycle threshold (Ct) values under 22 were identified as risk factors for
mortality. Analysis of the expression profiles of inflammatory pathway genes showed
that the greater the infection severity, the greater the activation of inflammatory
pathways, triggering the cytokine storm and downregulating anti-inflammatory
pathways. Viral genome analysis revealed the circulation of multiple lineages, such
as B.1, B.1.1.28, Alpha, and Gamma, suggesting that multiple introduction events
had occurred over time. This study’s findings help identify the specific
strains and increase our understanding of the true state of local health. In
addition, our data demonstrate that epidemiological and genomic surveillance
together can help formulate public health strategies to guide governmental actions.
Institute of Multidisciplinary Health, Federal University of Bahia, Rua
Hormindo Barros, 58, Candeias, Vitória da Conquista 45029-094, BA, Brazil
carolsaude5@hotmail.com; hellen.ufba@gmail.com; mylla.gomes@yahoo.com.br;
danieleleite.saude@gmail.com; pdbinfante@gmail.com; rosimaracaitite@gmail.com;
hegger.fritsch@gmail.com; marinacucco@hotmail.com; lucassantana864@gmail.com;
calinenovais@yahoo.com.br; rafaelasb@gmail.com; aline.amorim2011@hotmail.com;
analuiisanascimento@gmail.com; francellygoes@gmail.com;
daniellesoutomedeiros@gmail.com; mgalvao@ufba.br; smistro@ufba.br;
freiremelo@yahoo.com.br; taianat7@gmail.com; anamarcia@usp.br; joti@usp.br;
pablomaciel.farmacia@gmail.com; sandra.hp.oliveira@unesp.br;
luiz.alcantara@ioc.fiocruz.br; giovanetti.marta@gmail.com; luciane.tika@gmail.com;
vagner.fonseca@gmail.com; fernanda.khouri@hotmail.com;
guilhermebcampos@hotmail.com; lucasm@ufba.br 10.3390/v14112424 2022 14 11
- - 2424 -
Qu, Jipeng; Chen, Zhenyong; Wang, Bixia; Feng, Shiling; Tong, Zhaoguo; Chen, Tao;
Zhou, Lijun; Peng, Zhengsong; Ding, Chunbang Molecular Mechanisms Regulating the
Oil Biosynthesis in Olive (Olea europaea L.) Fruits Revealed by Transcriptomic
Analysis Agronomy EN Article oil biosynthesis; olive fruit;
transcriptomic analysis; fatty acid metabolism; transcription factor; co-expression
network As one of the most important crops for oil, olive (Olea europaea L.) is
well-known worldwide for its commercial product “virgin olive oil”
containing high-content fatty acids and many secondary metabolites. The molecular
mechanisms underlying the enhanced oil content in olive remain unclear. To further
investigate the molecular mechanisms of olive oil biosynthesis, we selected two
olive cultivars, i.e., Kalinjot (JZ) and Coratina (KLD), at three maturity stages
(MI-1, MI-3, and MI-6) for transcriptomic analysis based on Nanopore sequencing.
Significant differences were observed in oil content between JZ and KLD during
three maturity stages. Enrichment analysis revealed significant enrichment of
differentially expressed genes (DEGs) in metabolic pathways of photosynthesis,
amino acid biosynthesis, response to stress, and energy metabolism, in particular,
fatty acid metabolism. A total of 170 (31.54% of 539 genes involved in oil
synthesis) DEGs were further investigated based on expression analysis to identify
their molecular functions in oil biosynthesis in olive. A co-expression network
based on 714 transcription factors and their targeted genes in oil biosynthesis was
constructed. Our study provided novel experimental evidence to investigate the
molecular mechanisms of olive oil biosynthesis and to improve the breeding of olive
varieties with enhanced oil contents. College of Life Sciences, Sichuan
Agricultural University, Yaan 625014, China ququ8312@163.com;
chenzhenyong@cwnu.edu.cn; 696wbx@163.com; fengshilin@outlook.com;
olivetong@163.com; chentao293@163.com; zhoulijun@sicau.edu.cn; pzs8833@163.com;
dcb@sicau.edu.cn 10.3390/agronomy12112718 2022 12 11 - - 2718
-
Jiang, Rong-San; Shih, Chien-Hung; Jiang, Yu-Han; Hsieh, Han-Hsueh; Chiang, Yi-
Fang; Chuang, Han-Ni; Hsiao, Tzu-Hung Nasal Mycology of Chronic Rhinosinusitis
Revealed by Nanopore Sequencing Diagnostics EN Article chronic
rhinosinusitis; fungal culture; fungus; nanopore sequencingBackground: Nanopore
sequencing (NS) is a third-generation sequencing technology capable of generating
reads of long sequences. In this study, we used NS to investigate nasal mycology in
patients with chronic rhinosinusitis (CRS). Methods: Nasal cavities of 13 CRS
patients were individually irrigated with 20 mL of distilled water. The irrigant
was forcefully blown by the patient into a basin. The collected fluid was placed
into a centrifuge tube and processed using the method of Ponikau et al. The
collected specimens were used for traditional fungal culture and sequenced for
total DNA using NS. Results: Traditional fungal culture successfully grew fungi in
the specimens of 11 (84.6%) patients. Aspergillus sp. and Penicillium sp. were
found in four (30.8%) patients, Cladosporium sp. in three (23.1%) patients, and
Candida albicans, Mucor sp. and Chaetomium sp. in one patient. NS revealed fungi
abundance ranged from 81 to 2226, with the Shannon species diversity ranging from
1.094 to 1.683 at the genus level. Malassezia sp. was sequenced in 13 patients,
Aspergillus sp. in 12 (92.3%) patients, Candida albicans in 11 (84.6%) patients,
and Penicillium sp. in 10 (76.9%) patients. Conclusion: Our results showed that NS
was sensitive and fast in detecting nasal fungi in CRS patients. Department of
Medical Research, Taichung Veterans General Hospital, Taichung 40705, Taiwan
rsjiang@vghtc.gov.tw; vieri0305@gmail.com; pony810506@gmail.com;
hhhsieh1903@gmail.com; yvonnechiang9009@gmail.com; hannichuang@gmail.com;
thsiao@vghtc.gov.tw 10.3390/diagnostics12112735 2022 12 11 - -
2735 -
Rahube, Teddie; Cameron, Andrew; Lerminiaux, Nicole; Bhat, Supriya; Alexander,
Kathleen Globally Disseminated Multidrug Resistance Plasmids Revealed by
Complete Assembly of Multidrug Resistant Escherichia coli and
Klebsiella pneumoniae Genomes from Diarrheal Disease in Botswana Applied
Microbiology EN Article antimicrobial resistance; next generation
sequencing; plasmids; antibiotic resistance genes; BotswanaAntimicrobial resistance
is a disseminated global health challenge because many of the genes that cause
resistance can transfer horizontally between bacteria. Despite the central role of
extrachromosomal DNA elements called plasmids in driving the spread of resistance,
the detection and surveillance of plasmids remains a significant barrier in
molecular epidemiology. We assessed two DNA sequencing platforms alone and in
combination for laboratory diagnostics in Botswana by annotating antibiotic
resistance genes and plasmids in extensively drug resistant bacteria from diarrhea
in Botswana. Long-read Nanopore DNA sequencing and high accuracy basecalling
effectively estimated the architecture and gene content of three plasmids in
Escherichia coli HUM3355 and two plasmids in Klebsiella pneumoniae HUM7199.
Polishing the assemblies with Illumina reads increased base calling precision with
small improvements to gene prediction. All five plasmids encoded one or more
antibiotic resistance genes, usually within gene islands containing multiple
antibiotic and metal resistance genes, and four plasmids encoded genes associated
with conjugative transfer. Two plasmids were almost identical to antibiotic
resistance plasmids sequenced in Europe and North America from human infection and
a pig farm. These One Health connections demonstrate how low-, middle-, and high-
income countries collectively benefit from increased whole genome sequencing
capacity for surveillance and tracking of infectious diseases and antibiotic
resistance genes that can transfer between animal hosts and move across continents.
Department of Biological Sciences and Biotechnology, Faculty of Science,
Botswana International University of Science and Technology (BIUST), Private Bag
16, Palapye, Botswana rahubet@biust.ac.bw; andrew.cameron@uregina.ca;
lerminin@uregina.ca; supriya.bhat@uregina.ca; kathyalx@vt.edu
10.3390/applmicrobiol2040071 2022 2 4 - - 71 -
Wei, Wentao; Wang, Huiyuan; Liu, Xuqing; Kou, Wenjing; Liu, Ziqi; Wang, Huihui;
Yang, Yongkang; Zhao, Liangzhen; Zhang, Hangxiao; Liu, Bo; Ma, Xiangqing; Gu,
Lianfeng Transcriptome Profiling of Stem-Differentiating Xylem in Response to
Abiotic Stresses Based on Hybrid Sequencing in Cunninghamia lanceolata
 International Journal of Molecular Sciences EN Article Cunninghamia
lanceolate; stem-differentiating xylem; abiotic stress; nanopore long-read
sequencing; transcription factors Cunninghamia lanceolata (C. lanceolata) belongs
to Gymnospermae, which are fast-growing and have desirable wood properties.
However, C. lanceolata’s stress resistance is little understood. To unravel
the physiological and molecular regulation mechanisms under environmental stresses
in the typical gymnosperm species of C. lanceolata, three-year-old plants were
exposed to simulated drought stress (polyethylene glycol 8000), salicylic acid, and
cold treatment at 4 °C for 8 h, 32 h, and 56 h, respectively. Regarding the
physiological traits, we observed a decreased protein content and increased
peroxidase upon salicylic acid and polyethylene glycol treatment. Superoxide
dismutase activity either decreased or increased at first and then returned to
normal under the stresses. Regarding the molecular regulation, we used both
nanopore direct RNA sequencing and short-read sequencing to reveal a total of 5646
differentially expressed genes in response to different stresses, of which most had
functions in lignin catabolism, pectin catabolism, and xylan metabolism, indicating
that the development of stem-differentiating xylem was affected upon stress
treatment. Finally, we identified a total of 51 AP2/ERF, 29 NAC, and 37 WRKY
transcript factors in C. lanceolata. The expression of most of the NAC TFs
increased under cold stress, and the expression of most of the WRKY TFs increased
under cold and SA stress. These results revealed the transcriptomics responses in
C. lanceolata to short-term stresses under this study’s experimental
conditions and provide preliminary clues about stem-differentiating xylem changes
associated with different stresses. College of Forestry, Fujian Agriculture and
Forestry University, Fuzhou 350002, China 15571728818@163.com; whyfafu@163.com;
fafuxuqing@163.com; kwjfafu@163.com; lzq1402809112@163.com; whhfafu@163.com;
yyongkang2022@163.com; zhaoliangzhen80@163.com; zhanghx@fafu.edu.cn;
lboshandong@126.com; 000q131002@fafu.edu.cn; lfgu@fafu.edu.cn
10.3390/ijms232213986 2022 23 22 - - 13986 -
Martí-Carreras, Joan; Carrasco, Marina; Gómez-Ponce, Marcel; Noguera-Julián, Marc;
Fisa, Roser; Riera, Cristina; Alcover, Maria; Roura, Xavier; Ferrer, Lluís;
Francino, Olga Identification of Leishmania infantum Epidemiology, Drug
Resistance and Pathogenicity Biomarkers with Nanopore Sequencing Microorganisms
EN Communication Leishmania infantum; leishmaniosis; drug resistance;
treatment; nanopore sequencing; copy number variation; aneuploidy; maxicircle;
LeishGenApp The emergence of drug-resistant strains of the parasite Leishmania
infantum infecting dogs and humans represents an increasing threat. L. infantum
genomes are complex and unstable with extensive structural variations, ranging from
aneuploidies to multiple copy number variations (CNVs). These CNVs have recently
been validated as biomarkers of Leishmania concerning virulence, tissue tropism,
and drug resistance. As a proof-of-concept to develop a novel diagnosis platform
(LeishGenApp), four L. infantum samples from humans and dogs were nanopore
sequenced. Samples were epidemiologically typed within the Mediterranean L.
infantum group, identifying members of the JCP5 and non-JCP5 subgroups, using the
conserved region (CR) of the maxicircle kinetoplast. Aneuploidies were frequent and
heterogenous between samples, yet only chromosome 31 tetrasomy was common between
all the samples. A high frequency of aneuploidies was observed for samples with
long passage history (MHOM/TN/80/IPT-1), whereas fewer were detected for samples
maintained in vivo (MCRI/ES/2006/CATB033). Twenty-two genes were studied to
generate a genetic pharmacoresistance profile against miltefosine, allopurinol,
trivalent antimonials, amphotericin, and paromomycin. MHOM/TN/80/IPT-1 and
MCRI/ES/2006/CATB033 displayed a genetic profile with potential resistance against
miltefosine and allopurinol. Meanwhile, MHOM/ES/2016/CATB101 and
LCAN/ES/2020/CATB102 were identified as potentially resistant against paromomycin.
All four samples displayed a genetic profile for resistance against trivalent
antimonials. Overall, this proof-of-concept revealed the potential of nanopore
sequencing and LeishGenApp for the determination of epidemiological, drug
resistance, and pathogenicity biomarkers in L. infantum. Nano1Health S.L. (N1H),
Edifici EUREKA, Parc de Recerca UAB, Bellaterra, 08193 Barcelona, Spain
 jmarti@nano1health.com; mcarrasco@nano1health.com; mgomez@nano1health.com;
mnoguera@nano1health.com; rfisa@ub.edu; mcriera@ub.edu;
mmagdalenaalcoveramengual@ub.edu; xavier.roura@uab.cat; lluis.ferrer@uab.cat;
ofrancino@nano1health.com 10.3390/microorganisms10112256 2022 10 11
- - 2256 -
Viñes, Joaquim; Fàbregas, Norma; Pérez, Daniel; Cuscó, Anna; Fonticoba, Rocío;
Francino, Olga; Ferrer, Lluís; Migura-Garcia, Lourdes Concordance between
Antimicrobial Resistance Phenotype and Genotype of Staphylococcus pseudintermedius
from Healthy Dogs Antibiotics EN Article Staphylococcus pseudintermedius;
antibiotic resistance; nanopore sequencing; phenotype-genotype concordance; healthy
dog Staphylococcus pseudintermedius, a common commensal canine bacterium, is the
main cause of skin infections in dogs and is a potential zoonotic pathogen. The
emergence of methicillin-resistant S. pseudintermedius (MRSP) has compromised the
treatment of infections caused by these bacteria. In this study, we compared the
phenotypic results obtained by minimum inhibitory concentration (MICs) for 67 S.
pseudintermedius isolates from the skin of nine healthy dogs versus the genotypic
data obtained with Nanopore sequencing. A total of 17 antibiotic resistance genes
(ARGs) were detected among the isolates. A good correlation between phenotype and
genotype was observed for some antimicrobial classes, such as ciprofloxacin
(fluoroquinolone), macrolides, or tetracycline. However, for oxacillin (beta-
lactam) or aminoglycosides the correlation was low. Two antibiotic resistance genes
were located on plasmids integrated in the chromosome, and a third one was in a
circular plasmid. To our knowledge, this is the first study assessing the
correlation between phenotype and genotype regarding antimicrobial resistance of S.
pseudintermedius from healthy dogs using Nanopore sequencing technology.
Vetgenomics, Edifici EUREKA, PRUAB, Campus de la Universitat Autònoma de
Barcelona (UAB), Bellaterra, 08193 Barcelona, Spain
joaquim.vines@vetgenomics.com; norma.fabregas@vetgenomics.com;
daniel.perez.rodriguez@uab.cat; anna.cusco@vetgenomics.com;
rociofonticoba@yahoo.es; olga.francino@uab.cat; lluis.ferrer@uab.cat;
lourdes.migura@irta.cat 10.3390/antibiotics11111625 2022 11 11 - -
1625 -
Palombieri, Andrea; Fruci, Paola; Sarchese, Vittorio; Robetto, Serena; Orusa,
Riccardo; Arbuatti, Alessio; Martella, Vito; Di Martino, Barbara; Di Profio,
Federica Detection and Characterization of a Novel Picornavirus in European
Badger (Meles meles) Veterinary Sciences EN Communication
Picornaviridae; sakobuvirus; fecal virome; badger The recent development
of unbiased metagenomic next-generation sequencing has provided a richer view of
the wild animal virome making it necessary to expand the knowledge about virus
diversity in wildlife, as well as to monitor their potential transmission to
domestic animals or humans. In the present study, by screening collections of
enteric specimens from wild animals, a novel picornavirus was identified in the
intestinal content of a badger (Meles meles). By enrichment with a sequence-
independent single-primer amplification (SISPA) approach and deep sequencing with
Oxford Nanopore Technologies (ONT) platform, the genome sequence of a novel
picornavirus strain, Badger/3A-2019/ITA, was reconstructed. On comparison based on
the polyprotein sequences, the virus was distantly related (58.7% and 59.7%
sequence identity at the nucleotide and amino acid level, respectively) to the
feline picornavirus strain FFUP1, identified in 2012 in Portugal and classified
into genus Sakobovirus within the species Sakobuvirus A. Upon phylogenetic,
pairwise homology, and distance analyses performed on the P1, 2Chel, 3Cpro, and
3Dpol proteins and the complete genomic sequence, the badger picornavirus may be
considered a member of a new sakobuvirus species, which we propose as Sakobuvirus
B. Department of Veterinary Medicine, Università degli Studi di Teramo, 64100
Teramo, Italy apalombieri@unite.it; pfruci@unite.it; vsarchese@unite.it;
serena.robetto@izsto.it; riccardo.orusa@izsto.it; aarbuatti@unite.it;
vito.martella@uniba.it; bdimartino@unite.it; fdiprofio@unite.it
10.3390/vetsci9110645 2022 9 11 - - 645 -
Muñoz-Barrera, Adrián; Rubio-Rodríguez, Luis; Díaz-de Usera, Ana; Jáspez, David;
Lorenzo-Salazar, José; González-Montelongo, Rafaela; García-Olivares, Víctor;
Flores, Carlos From Samples to Germline and Somatic Sequence Variation: A Focus
on Next-Generation Sequencing in Melanoma Research Life EN Review cancer
genomics; melanoma; next-generation sequencing; third-generation sequencing;
nanopore; bioinformatic workflows; pipeline; clinical genomics; personalized
medicine Next-generation sequencing (NGS) applications have flourished in the
last decade, permitting the identification of cancer driver genes and profoundly
expanding the possibilities of genomic studies of cancer, including melanoma. Here
we aimed to present a technical review across many of the methodological approaches
brought by the use of NGS applications with a focus on assessing germline and
somatic sequence variation. We provide cautionary notes and discuss key technical
details involved in library preparation, the most common problems with the samples,
and guidance to circumvent them. We also provide an overview of the sequence-based
methods for cancer genomics, exposing the pros and cons of targeted sequencing vs.
exome or whole-genome sequencing (WGS), the fundamentals of the most common
commercial platforms, and a comparison of throughputs and key applications. Details
of the steps and the main software involved in the bioinformatics processing of the
sequencing results, from preprocessing to variant prioritization and filtering, are
also provided in the context of the full spectrum of genetic variation (SNVs,
indels, CNVs, structural variation, and gene fusions). Finally, we put the emphasis
on selected bioinformatic pipelines behind (a) short-read WGS identification of
small germline and somatic variants, (b) detection of gene fusions from
transcriptomes, and (c) de novo assembly of genomes from long-read WGS data.
Overall, we provide comprehensive guidance across the main methodological
procedures involved in obtaining sequencing results for the most common short- and
long-read NGS platforms, highlighting key applications in melanoma research.
Genomics Division, Instituto Tecnológico y de Energías Renovables (ITER),
38600 Santa Cruz de Tenerife, Spain amunoz@iter.es; lrubio@iter.es;
anadidu07@gmail.com; djaspez@iter.es; jlorenzo@iter.es;
rgonzalezmontelongo@iter.es; vgarcia@iter.es; cflores@ull.edu.es
10.3390/life12111939 2022 12 11 - - 1939 -
Bologa, Alexandru; Stoica, Ileana; Ratiu, Attila; Constantin, Nicoleta; Ecovoiu,
Alexandru ONT-Based Alternative Assemblies Impact on the Annotations of Unique
versus Repetitive Features in the Genome of a Romanian Strain of Drosophila
melanogaster International Journal of Molecular Sciences EN Article
Drosophila melanogaster; nanopore sequencing; MinION; ONT; de novo genome
assembly; natural transposons To date, different strategies of whole-genome
sequencing (WGS) have been developed in order to understand the genome structure
and functions. However, the analysis of genomic sequences obtained from natural
populations is challenging and the biological interpretation of sequencing data
remains the main issue. The MinION device developed by Oxford Nanopore Technologies
(ONT) is able to generate long reads with minimal costs and time requirements.
These valuable assets qualify it as a suitable method for performing WGS,
especially in small laboratories. The long reads resulted using this sequencing
approach can cover large structural variants and repetitive sequences commonly
present in the genomes of eukaryotes. Using MinION, we performed two WGS
assessments of a Romanian local strain of Drosophila melanogaster, referred to as
Horezu_LaPeri (Horezu). In total, 1,317,857 reads with a size of 8.9 gigabytes (Gb)
were generated. Canu and Flye de novo assembly tools were employed to obtain four
distinct assemblies with both unfiltered and filtered reads, achieving maximum
reference genome coverages of 94.8% (Canu) and 91.4% (Flye). In order to test the
quality of these assemblies, we performed a two-step evaluation. Firstly, we
considered the BUSCO scores and inquired for a supplemental set of genes using
BLAST. Subsequently, we appraised the total content of natural transposons (NTs)
relative to the reference genome (ISO1 strain) and mapped the mdg1 retroelement as
a resolution assayer. Our results reveal that filtered data provide only slightly
enhanced results when considering genes identification, but the use of unfiltered
data had a consistent positive impact on the global evaluation of the NTs content.
Our comparative studies also revealed differences between Flye and Canu assemblies
regarding the annotation of unique versus repetitive genomic features. In our
hands, Flye proved to be moderately better for gene identification, while Canu
clearly outperformed Flye for NTs analysis. Data concerning the NTs content were
compared to those obtained with ONT for the D. melanogaster ISO1 strain, revealing
that our strategy conducted to better results. Additionally, the parameters of our
ONT reads and assemblies are similar to those reported for ONT experiments
performed on various model organisms, revealing that our assembly data are
appropriate for a proficient annotation of the Horezu genome. Department of
Genetics, Faculty of Biology, University of Bucharest, 060101 Bucharest, Romania
alexandru.bologa@drd.unibuc.ro; ileana.stoica@bio.unibuc.ro;
attila.ratiu@bio.unibuc.ro; constantin_nicoleta-denisa@s.bio.unibuc.ro;
alexandru.ecovoiu@bio.unibuc.ro 10.3390/ijms232314892 2022 23 23 -
- 14892 -
Kovács, Árpád; Némethi, Zaránd; Abonyi, Tünde; Fekete, György; Kovács, Gábor
Enhancing Molecular Testing for Effective Delivery of Actionable Gene
Diagnostics Bioengineering EN Review actionable genetic diagnosis;
nanopore sequencing; long-read sequencing; complex structural variants; single
cells; genetic counselling There is a deep need to navigate within our genomic
data to find, understand and pave the way for disease-specific treatments, as the
clinical diagnostic journey provides only limited guidance. The human genome is
enclosed in every nucleated cell, and yet at the single-cell resolution many
unanswered questions remain, as most of the sequencing techniques use a bulk
approach. Therefore, heterogeneity, mosaicism and many complex structural variants
remain partially uncovered. As a conceptual approach, nanopore-based sequencing
holds the promise of being a single-molecule-based, long-read and high-resolution
technique, with the ability of uncovering the nucleic acid sequence and methylation
almost in real time. A key limiting factor of current clinical genetics is the
deciphering of key disease-causing genomic sequences. As the technological
revolution is expanding regarding genetic data, the interpretation of
genotype–phenotype correlations should be made with fine caution, as more and
more evidence points toward the presence of more than one pathogenic variant acting
together as a result of intergenic interplay in the background of a certain
phenotype observed in a patient. This is in conjunction with the observation that
many inheritable disorders manifest in a phenotypic spectrum, even in an intra-
familial way. In the present review, we summarized the relevant data on nanopore
sequencing regarding clinical genomics as well as highlighted the importance and
content of pre-test and post-test genetic counselling, yielding a complex approach
to phenotype-driven molecular diagnosis. This should significantly lower the time-
to-right diagnosis as well lower the time required to complete a currently
incomplete genotype–phenotype axis, which will boost the chance of
establishing a new actionable diagnosis followed by therapeutical approach. 2nd
Department of Paediatrics, Semmelweis University, Üllői út 26, 1085 Budapest,
Hungary kovacs.arpad@med.semmelweis-univ.hu; nemethi.zarand@med.semmelweis-
univ.hu; abonyi.tunde@med.semmelweis-univ.hu; fekete.gyorgy@med.semmelweis-univ.hu;
kovacs.gabor1@med.semmelweis-univ.hu 10.3390/bioengineering9120745 2022 9
12 - - 745 -
Kolesnikova, Tatyana; Klenov, Mikhail; Nokhova, Alina; Lavrov, Sergey; Pokholkova,
Galina; Schubert, Veit; Maltseva, Svetlana; Cook, Kevin; Dixon, Michael; Zhimulev,
Igor A Spontaneous Inversion of the X Chromosome Heterochromatin Provides a Tool
for Studying the Structure and Activity of the Nucleolus in Drosophila melanogaster
Cells EN Article heterochromatin; nucleolus; inversion; Rif1;
Drosophila melanogaster; polytene chromosomes; underreplication; In(1)sc8 The
pericentromeric heterochromatin is largely composed of repetitive sequences, making
it difficult to analyze with standard molecular biological methods. At the same
time, it carries many functional elements with poorly understood mechanisms of
action. The search for new experimental models for the analysis of heterochromatin
is an urgent task. In this work, we used the Rif1 mutation, which suppresses the
underreplication of all types of repeated sequences, to analyze heterochromatin
regions in polytene chromosomes of Drosophila melanogaster. In the Rif1 background,
we discovered and described in detail a new inversion, In(1)19EHet, which arose on
a chromosome already carrying the In(1)sc8 inversion and transferred a large part
of X chromosome heterochromatin, including the nucleolar organizer to a new
euchromatic environment. Using nanopore sequencing and FISH, we have identified the
eu- and heterochromatin breakpoints of In(1)19EHet. The combination of the new
inversion and the Rif1 mutation provides a promising tool for studies of X
chromosome heterochromatin structure, nucleolar organization, and the nucleolar
dominance phenomenon. In particular, we found that, with the complete
polytenization of rDNA repeats, the nucleolus consists of a cloud-like structure
corresponding to the classical nucleolus of polytene chromosomes, as well as an
unusual intrachromosomal structure containing alternating transcriptionally active
and inactive regions. Institute of Molecular and Cellular Biology SB RAS, 630090
Novosibirsk, Russia kolesnikova@mcb.nsc.ru; klenov@img.ras.ru; alina-
nohova@mail.ru; slavrov@img.ras.ru; galina@mcb.nsc.ru; schubertv@ipk-
gatersleben.de; svm31783@gmail.com; kercook@indiana.edu; midixon@indiana.edu;
zhimulev@mcb.nsc.ru 10.3390/cells11233872 2022 11 23 - - 3872
-
Dieng, Idrissa; Barry, Mamadou; Talla, Cheikh; Sow, Bocar; Faye, Oumar; Diagne,
Moussa; Sene, Ousseynou; Ndiaye, Oumar; Diop, Boly; Diagne, Cheikh; Fall, Gamou;
Sall, Amadou; Loucoubar, Cheikh; Faye, Ousmane Analysis of a Dengue Virus Outbreak
in Rosso, Senegal 2021 Tropical Medicine and Infectious Disease EN Article
DENV-1; Rosso; NS1 RDTs; outbreak response; serotype replacement; re-
introduction Senegal is hyperendemic for dengue. Since 2017, outbreaks have
been noticed annually in many regions around the country, marked by the co-
circulation of DENV1-3. On 8 October 2021, a Dengue virus outbreak in the Rosso
health post (sentinel site of the syndromic surveillance network) located in the
north of the country was notified to the WHO Collaborating Center for arboviruses
and hemorrhagic fever viruses at Institut Pasteur de Dakar. A multidisciplinary
team was then sent for epidemiological and virologic investigations. This study
describes the results from investigations during an outbreak in Senegal using a
rapid diagnostic test (RDT) for the combined detection of dengue virus non-
structural protein 1 (NS1) and IgM/IgG. For confirmation, samples were also tested
by real-time RT-PCR and IgM ELISA at the reference lab in Dakar. qRT-PCR positive
samples were subjected to whole genome sequencing using nanopore technology.
Virologic analysis scored 102 positives cases (RT-PCR, NS1 antigen detection and/or
IgM) out of 173 enrolled patients; interestingly, virus serotyping showed that the
outbreak was caused by the DENV-1, a serotype different from DENV-2 involved during
the outbreak in Rosso three years earlier, indicating a serotype replacement.
Nearly all field-tested NS1 positives samples were confirmed by qRT-PCR with a
concordance of 92.3%. Whole genome sequencing and phylogenetic analysis of strains
suggested a re-introduction in Rosso of a DENV-1 strain different to the one
responsible for the outbreak in the Louga area five years before. Findings call for
improved dengue virus surveillance in Senegal, with a wide deployment of DENV
antigenic tests, which allow easy on-site diagnosis of suspected cases and early
detection of outbreaks. This work highlights the need for continuous monitoring of
circulating serotypes which is crucial for a better understanding of viral
epidemiology around the country. Arboviruses and Haemorrhagic Fever Viruses
Unit, Virology Department, Institute Pasteur de Dakar, Dakar 220, Senegal
idrissa.dieng@pasteur.sn; aliou.barry@pasteur.sn; cheikh.talla@pasteur.sn;
bocar.sow@pasteur.sn; oumar.faye@pasteur.sn; moussamoise.diagne@pasteur.sn;
senesenely@gmail.com; oumar.ndiaye@pasteur.sn; diopboly@gmail.com;
cheikhtidiane.diagne@pasteur.sn; gamou.fall@pasteur.sn; amadou.sall@pasteur.sn;
cheikh.loucoubar@pasteur.sn; ousmane.faye@pasteur.sn 10.3390/tropicalmed7120420
2022 7 12 - - 420 -
Schulz, Ansgar; Sadeghi, Balal; Stoek, Franziska; King, Jacqueline; Fischer,
Kerstin; Pohlmann, Anne; Eiden, Martin; Groschup, Martin Whole-Genome Sequencing
of Six Neglected Arboviruses Circulating in Africa Using Sequence-Independent
Single Primer Amplification (SISPA) and MinION Nanopore Technologies Pathogens
EN Article MinION sequencing; SISPA; arboviruses; Africa On the
African continent, a large number of arthropod-borne viruses (arboviruses) with
zoonotic potential have been described, and yet little is known of most of these
pathogens, including their actual distribution or genetic diversity. In this study,
we evaluated as a proof-of-concept the effectiveness of the nonspecific sequencing
technique sequence-independent single primer amplification (SISPA) on third-
generation sequencing techniques (MinION sequencing, Oxford Nanopore Technologies,
Oxford, UK) by comparing the sequencing results from six different samples of
arboviruses known to be circulating in Africa (Crimean–Congo hemorrhagic
fever virus (CCHFV), Rift Valley fever virus (RVFV), Dugbe virus (DUGV), Nairobi
sheep disease virus (NSDV), Middleburg virus (MIDV) and Wesselsbron virus (WSLV)).
All sequenced samples were derived either from previous field studies or animal
infection trials. Using this approach, we were able to generate complete genomes
for all six viruses without the need for virus-specific whole-genome PCRs. Higher
Cq values in diagnostic RT-qPCRs and the origin of the samples (from cell culture
or animal origin) along with their quality were found to be factors affecting the
success of the sequencing run. The results of this study may stimulate the use of
metagenomic sequencing approaches, contributing to a better understanding of the
genetic diversity of neglected arboviruses. Institute of Novel and Emerging
Infectious Diseases, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems,
Germany ansgar.schulz@fli.de; balal.sadeghi@fli.de; franziska.stoek@fli.de;
jacqueline.king@fli.de; kerstin.fischer@fli.de; anne.pohlmann@fli.de;
martin.eiden@fli.de; martin.groschup@fli.de 10.3390/pathogens11121502 2022
11 12 - - 1502 -
Skowronek, Dariush; Pilz, Robin; Bonde, Loisa; Schamuhn, Ole; Feldmann, Janne;
Hoffjan, Sabine; Much, Christiane; Felbor, Ute; Rath, Matthias Cas9-Mediated
Nanopore Sequencing Enables Precise Characterization of Structural Variants in CCM
Genes International Journal of Molecular Sciences EN Article nanopore
sequencing; long-read sequencing; CRISPR/Cas9; copy number variants; cerebral
cavernous malformations; structural variants Deletions in the CCM1, CCM2, and
CCM3 genes are a common cause of familial cerebral cavernous malformations (CCMs).
In current molecular genetic laboratories, targeted next-generation sequencing or
multiplex ligation-dependent probe amplification are mostly used to identify copy
number variants (CNVs). However, both techniques are limited in their ability to
specify the breakpoints of CNVs and identify complex structural variants (SVs). To
overcome these constraints, we established a targeted Cas9-mediated nanopore
sequencing approach for CNV detection with single nucleotide resolution. Using a
MinION device, we achieved complete coverage for the CCM genes and determined the
exact size of CNVs in positive controls. Long-read sequencing for a CCM1 and CCM2
CNV revealed that the adjacent ANKIB1 and NACAD genes were also partially or
completely deleted. In addition, an interchromosomal insertion and an inversion in
CCM2 were reliably re-identified by long-read sequencing. The refinement of CNV
breakpoints by long-read sequencing enabled fast and inexpensive PCR-based variant
confirmation, which is highly desirable to reduce costs in subsequent family
analyses. In conclusion, Cas9-mediated nanopore sequencing is a cost-effective and
flexible tool for molecular genetic diagnostics which can be easily adapted to
various target regions. Department of Human Genetics, University Medicine
Greifswald and Interfaculty Institute of Genetics and Functional Genomics,
University of Greifswald, 17475 Greifswald, Germany dariush.skowronek@med.uni-
greifswald.de; robinalexander.pilz@med.uni-greifswald.de; loisadana.bonde@med.uni-
greifswald.de; ole.schamuhn@stud.uni-greifswald.de; s-jafeld@uni-greifswald.de;
sabine.hoffjan@ruhr-uni-bochum.de; christiane.much@med.uni-greifswald.de;
ute.felbor@med.uni-greifswald.de; matthias.rath@med.uni-greifswald.de
10.3390/ijms232415639 2022 23 24 - - 15639 -
Wang, Yupeng; Yuan, Jianxuan; Deng, Haofeng; Zhang, Ziang; Ma, Qianli; Wu, Lingzhi;
Weng, Lixing Procedural Data Processing for Single-Molecule Identification by
Nanopore Sensors Biosensors EN Article current pulse; signal
identification; nanopore; single molecular event Nanopores are promising
single-molecule sensing devices that have been successfully used for DNA
sequencing, protein identification, as well as virus/particles detection. It is
important to understand and characterize the current pulses collected by nanopore
sensors, which imply the associated information of the analytes, including the
size, structure, and surface charge. Therefore, a signal processing program, based
on the MATLAB platform, was designed to characterize the ionic current signals of
nanopore measurements. In a movable data window, the selected current segment was
analyzed by the adaptive thresholds and corrected by multi-functions to reduce the
noise obstruction of pulse signals. Accordingly, a set of single molecular events
was identified, and the abundant information of current signals with the dwell
time, amplitude, and current pulse area was exported for quantitative analysis. The
program contributes to the efficient and fast processing of nanopore signals with a
high signal-to-noise ratio, which promotes the development of the nanopore sensing
devices in various fields of diagnosis systems and precision medicine. School of
Materials Science & Engineering, Nanjing University of Posts and
Telecommunications, Nanjing 210023, China wangyupeng-x@foxmail.com;
yuanjx1998@126.com; 18762842307@163.com; zza18052256609@163.com; maql@njupt.edu.cn;
wulz@njupt.edu.cn; lxweng@njupt.edu.cn 10.3390/bios12121152 2022 12 12
- - 1152 -
Salakhov, Ramil; Golubenko, Maria; Valiakhmetov, Nail; Pavlyukova, Elena; Zarubin,
Aleksei; Babushkina, Nadezhda; Kucher, Aksana; Sleptcov, Aleksei; Nazarenko, Maria
Application of Long-Read Nanopore Sequencing to the Search for Mutations in
Hypertrophic Cardiomyopathy International Journal of Molecular Sciences EN
Article hypertrophic cardiomyopathy; long-read sequencing; Oxford
Nanopore; sarcomeric protein genes Increasing evidence suggests that both coding
and non-coding regions of sarcomeric protein genes can contribute to hypertrophic
cardiomyopathy (HCM). Here, we introduce an experimental workflow (tested on four
patients) for complete sequencing of the most common HCM genes (MYBPC3, MYH7, TPM1,
TNNT2, and TNNI3) via long-range PCR, Oxford Nanopore Technology (ONT) sequencing,
and bioinformatic analysis. We applied Illumina and Sanger sequencing to validate
the results, FastQC, Qualimap, and MultiQC for quality evaluations, MiniMap2 to
align data, Clair3 to call and phase variants, and Annovar’s tools and CADD
to assess pathogenicity of variants. We could not amplify the region encompassing
exons 6–12 of MYBPC3. A higher sequencing error rate was observed with ONT
(6.86–6.92%) than with Illumina technology (1.14–1.35%), mostly for
small indels. Pathogenic variant p.Gln1233Ter and benign polymorphism p.Arg326Gln
in MYBPC3 in a heterozygous state were found in one patient. We demonstrated the
ability of ONT to phase single-nucleotide variants, enabling direct haplotype
determination for genes TNNT2 and TPM1. These findings highlight the importance of
long-range PCR efficiency, as well as lower accuracy of variant calling by ONT than
by Illumina technology; these differences should be clarified prior to clinical
application of the ONT method. Research Institute of Medical Genetics, Tomsk
National Research Medical Center, Russian Academy of Sciences, 634050 Tomsk, Russia
ramil.salakhov@medgenetics.ru; maria.golubenko@medgenetics.ru;
nail.valiakhmetov@medgenetics.ru; pavluk@cardio-tomsk.ru;
aleksei.zarubin@medgenetics.ru; nad.babushkina@medgenetics.ru;
aksana.kucher@medgenetics.ru; alexei.sleptcov@medgenetics.ru;
maria.nazarenko@medgenetics.ru 10.3390/ijms232415845 2022 23 24 -
- 15845 -
Nelson, Theodore; Ghosh, Sankar; Postler, Thomas L-RAPiT: A Cloud-Based
Computing Pipeline for the Analysis of Long-Read RNA Sequencing Data
International Journal of Molecular Sciences EN Article LINC00173;
alternative splicing; bioinformatics; computational genomics; next-generation
sequencing; software; PacBio; Oxford Nanopore; long-read sequencing; RNA
sequencing; RNA-seq Long-read sequencing (LRS) has been adopted to meet a wide
variety of research needs, ranging from the construction of novel transcriptome
annotations to the rapid identification of emerging virus variants. Amongst other
advantages, LRS preserves more information about RNA at the transcript level than
conventional high-throughput sequencing, including far more accurate and
quantitative records of splicing patterns. New studies with LRS datasets are being
published at an exponential rate, generating a vast reservoir of information that
can be leveraged to address a host of different research questions. However, mining
such publicly available data in a tailored fashion is currently not easy, as the
available software tools typically require familiarity with the command-line
interface, which constitutes a significant obstacle to many researchers.
Additionally, different research groups utilize different software packages to
perform LRS analysis, which often prevents a direct comparison of published results
across different studies. To address these challenges, we have developed the Long-
Read Analysis Pipeline for Transcriptomics (L-RAPiT), a user-friendly, free
pipeline requiring no dedicated computational resources or bioinformatics
expertise. L-RAPiT can be implemented directly through Google Colaboratory, a
system based on the open-source Jupyter notebook environment, and allows for the
direct analysis of transcriptomic reads from Oxford Nanopore and PacBio LRS
machines. This new pipeline enables the rapid, convenient, and standardized
analysis of publicly available or newly generated LRS datasets. Department of
Microbiology & Immunology, Vagelos College of Physicians & Surgeons,
Columbia University Irving Medical Center, New York, NY 10032, USA
tmn2126@columbia.edu; sg2715@cumc.columbia.edu; tp2405@cumc.columbia.edu
10.3390/ijms232415851 2022 23 24 - - 15851 -
Capraru, Ionut; Romanescu, Mirabela; Anghel, Flavia; Oancea, Cristian; Marian,
Catalin; Sirbu, Ioan; Chis, Aimee; Ciordas, Paula Identification of Genomic
Variants of SARS-CoV-2 Using Nanopore Sequencing Medicina EN Article
SARS-CoV-2; sequencing; Nanopore; MinION; Ion TorrentBackground and
Objectives: SARS-CoV-2 is the first global threat and life-changing event of the
twenty-first century. Although efficient treatments and vaccines have been
developed, due to the virus’s ability to mutate in key regions of the genome,
whole viral genome sequencing is needed for efficient monitoring, evaluation of the
spread, and even the adjustment of the molecular diagnostic assays. Materials and
Methods: In this study, Nanopore and Ion Torrent sequencing technologies were used
to detect the main SARS-CoV-2 circulating strains in Timis County, Romania, between
February 2021 and May 2022. Results: We identified 22 virus lineages belonging to
seven clades: 20A, 20I (Alpha, V1), 21B (Kappa), 21I (Delta), 21J (Delta), 21K
(Omicron), and 21L (Omicron). Conclusions: Results obtained with both methods are
comparable, and we confirm the utility of Nanopore sequencing in large-scale
epidemiological surveillance due to the lower cost and reduced time for library
preparation. Discipline of Epidemiology, “Victor Babes” University of Medicine
and Pharmacy, 300041 Timișoara, Romania capraru.ionut@umft.ro;
mirabela.romanescu@umft.ro; flaviaanghel7@gmail.com; oancea@umft.ro;
cmarian@umft.ro; ovidiu.sirbu@umft.ro; chis.aimee@umft.ro; paula.muntean@umft.ro
10.3390/medicina58121841 2022 58 12 - - 1841 -
Kiel, Annika; Creutz, Ines; Rückert, Christian; Kaltschmidt, Bernhard; Hütten,
Andreas; Niehaus, Karsten; Busche, Tobias; Kaltschmidt, Barbara; Kaltschmidt,
Christian Genome-Based Analysis of Virulence Factors and Biofilm Formation in
Novel P. aeruginosa Strains Isolated from Household Appliances Microorganisms
EN Article Pseudomonas aeruginosa; biofilm; virulence factors;
household appliances; whole genome sequencing; prophages In household washing
machines, opportunistic pathogens such as Pseudomonas aeruginosa are present, which
represent the household as a possible reservoir for clinical pathogens. Here, four
novel P. aeruginosa strains, isolated from different sites of household appliances,
were investigated regarding their biofilm formation. Only two isolates showed
strong surface-adhered biofilm formation. In consequence of these phenotypic
differences, we performed whole genome sequencing using Oxford Nanopore Technology
together with Illumina MiSeq. Whole genome data were screened for the prevalence of
285 virulence- and biofilm-associated genes as well as for prophages. Linking
biofilm phenotypes and parallelly appearing gene compositions, we assume a
relevancy of the las quorum sensing system and the phage-encoded bacteriophage
control infection gene bci, which was found on integrated phi297 DNA in all
biofilm-forming isolates. Additionally, only the isolates revealing strong biofilm
formation harbored the ϕCTX-like prophage Dobby, implicating a role of this
prophage on biofilm formation. Investigations on clinically relevant pathogens
within household appliances emphasize their adaptability to harsh environments,
with high concentrations of detergents, providing greater insights into
pathogenicity and underlying mechanisms. This in turn opens the possibility to map
and characterize potentially relevant strains even before they appear as pathogens
in society. Department of Cell Biology, Faculty of Biology, Bielefeld University,
33615 Bielefeld, Germany annika.kiel@uni-bielefeld.de; ines.creutz@uni-
bielefeld.de; christian.rueckert@cebitec.uni-bielefeld.de; b.kaltschmidt@uni-
bielefeld.de; andreas.huetten@uni-bielefeld.de; kniehaus@cebitec.uni-bielefeld.de;
tbusche@cebitec.uni-bielefeld.de; barbara.kaltschmidt@uni-bielefeld.de;
c.kaltschmidt@uni-bielefeld.de 10.3390/microorganisms10122508 2022 10
12 - - 2508 -
Kirov, Ilya; Merkulov, Pavel; Polkhovskaya, Ekaterina; Konstantinov, Zakhar;
Kazancev, Mikhail; Saenko, Ksenia; Polkhovskiy, Alexander; Dudnikov, Maxim;
Garibyan, Tsovinar; Demurin, Yakov; Soloviev, Alexander Epigenetic Stress and
Long-Read cDNA Sequencing of Sunflower (Helianthus annuus L.) Revealed the Origin
of the Plant Retrotranscriptome Plants EN Article mobile elements;
LTR retrotransposons; Nanopore sequencing; sunflower; epigenetic stress; GAG
Transposable elements (TEs) contribute not only to genome diversity but also
to transcriptome diversity in plants. To unravel the sources of LTR retrotransposon
(RTE) transcripts in sunflower, we exploited a recently developed transposon
activation method (‘TEgenesis’) along with long-read cDNA Nanopore
sequencing. This approach allows for the identification of 56 RTE transcripts from
different genomic loci including full-length and non-autonomous RTEs. Using the
mobilome analysis, we provided a new set of expressed and transpositional active
sunflower RTEs for future studies. Among them, a Ty3/Gypsy RTE called SUNTY3
exhibited ongoing transposition activity, as detected by eccDNA analysis. We showed
that the sunflower genome contains a diverse set of non-autonomous RTEs encoding a
single RTE protein, including the previously described TR-GAG (terminal repeat with
the GAG domain) as well as new categories, TR-RT-RH, TR-RH, and TR-INT-RT. Our
results demonstrate that 40% of the loci for RTE-related transcripts (nonLTR-RTEs)
lack their LTR sequences and resemble conventional eucaryotic genes encoding RTE-
related proteins with unknown functions. It was evident based on phylogenetic
analysis that three nonLTR-RTEs encode GAG (HadGAG1-3) fused to a host protein.
These HadGAG proteins have homologs found in other plant species, potentially
indicating GAG domestication. Ultimately, we found that the sunflower
retrotranscriptome originated from the transcription of active RTEs, non-autonomous
RTEs, and gene-like RTE transcripts, including those encoding domesticated
proteins. All-Russia Research Institute of Agricultural Biotechnology,
Timiryazevskaya Str. 42, 127550 Moscow, Russia kirovez@gmail.com;
paulmerkulov97@gmail.com; eynzeynkreyn@gmail.com; zakhar.konstantinov@mail.ru;
irelanddets@gmail.com; saenkok1997@yandex.ru; polkhovsky.a.w@gmail.com;
max.dudnikov.07@gmail.com; tsovinar1980@mail.ru; genetic@vniimk.ru;
a.soloviev70@gmail.com 10.3390/plants11243579 2022 11 24 - - 3579
-
Sharma, Chayan; Khurana, Sumeeta; Arora, Amit; Bhatia, Alka; Gupta, Amit An
Insight into the Genome of Pathogenic and Non-Pathogenic Acanthamoeba Pathogens
EN Article Acanthamoeba; granulomatous amoebic encephalitis; hybrid
assembly; keratitis; next-generation sequencing Background: Acanthamoeba are
amphizoic amoeba majorly responsible for causing Acanthamoeba keratitis (AK) and
Granulomatous amoebic encephalitis (GAE). Despite its ubiquitous nature, the
frequency of infections is not high, probably due to the existence of non-
pathogenic isolates. The whole-genome sequencing and an annotated genome assembly
can unravel the biological functions and help in identifying probable genes related
to pathogenicity. Methods: Illumina and Nanopore sequencing were performed for
keratitis, encephalitis, and non-pathogenic environmental isolates. Hybrid assembly
was prepared for the AK and GAE isolates, while only the Illumina reads were
utilized for a non-pathogenic environmental isolate. Protein coding genes were
identified using the GeneMark-ES program and BLASTx module of Diamond used for gene
prediction. Additionally, the Kyoto Encyclopedia of Genes and Genomes annotation
and cluster of orthologous group’s annotation using RPS-blast against the CDD
database was performed. The subsequent data analysis and validation will help
identify probable pathogenic genes. Results: The genome assemblies of 9.67, 8.34,
and 8.89 GBs were reported for GAE, AK, and non-pathogenic isolate, respectively.
KEGG reported 22,946 in GAE, 24,231 in keratitis, and 9367 genes in the
environmental isolate. The COG annotation revealed 3232 in GAE, 3403 in keratitis,
and 1314 genes in the non-pathogenic isolate. Conclusion: The present study has
attempted to generate de novo hybrid genome assemblies of Acanthamoeba that would
help decode the genome of free-living amoeba and will provide genomic data for a
better understanding of virulence-related factors. Department of Medical
Parasitology, Postgraduate Institute of Medical Education & Research,
Chandigarh 160012, India chayansharma_1993@yahoo.com;
sumeetakhurana@hotmail.com; aarora.pgi@gmail.com; alkabhatia@ymail.com;
amitguptaeye@gmail.com 10.3390/pathogens11121558 2022 11 12 - -
1558 -
Pheiffer, Fazlin; Schneider, Yannik; Hansen, Espen; Andersen, Jeanette; Isaksson,
Johan; Busche, Tobias; Rückert, Christian; Kalinowski, Jörn; Zyl, Leonardo;
Trindade, Marla Bioassay-Guided Fractionation Leads to the Detection of Cholic
Acid Generated by the Rare Thalassomonas sp. Marine Drugs EN Article
Thalassomonas; cholic acid; bioactivity; genome mining; bile acid; bacterial
natural products; homoserine lactone acylase Bacterial symbionts of marine
invertebrates are rich sources of novel, pharmaceutically relevant natural products
that could become leads in combatting multidrug-resistant pathogens and treating
disease. In this study, the bioactive potential of the marine invertebrate symbiont
Thalassomonas actiniarum was investigated. Bioactivity screening of the strain
revealed Gram-positive specific antibacterial activity as well as cytotoxic
activity against a human melanoma cell line (A2058). The dereplication of the
active fraction using HPLC-MS led to the isolation and structural elucidation of
cholic acid and 3-oxo cholic acid. T. actiniarum is one of three type species
belonging to the genus Thalassomonas. The ability to generate cholic acid was
assessed for all three species using thin-layer chromatography and was confirmed by
LC-MS. The re-sequencing of all three Thalassomonas type species using long-read
Oxford Nanopore Technology (ONT) and Illumina data produced complete genomes,
enabling the bioinformatic assessment of the ability of the strains to produce
cholic acid. Although a complete biosynthetic pathway for cholic acid synthesis in
this genus could not be determined based on sequence-based homology searches, the
identification of putative penicillin or homoserine lactone acylases in all three
species suggests a mechanism for the hydrolysis of conjugated bile acids present in
the growth medium, resulting in the generation of cholic acid and 3-oxo cholic
acid. With little known currently about the bioactivities of this genus, this study
serves as the foundation for future investigations into their bioactive potential
as well as the potential ecological role of bile acid transformation, sterol
modification and quorum quenching by Thalassomonas sp. in the marine environment.
Institute for Microbial Biotechnology and Metagenomics (IMBM), Department of
Biotechnology, University of the Western Cape, Robert Sobukwe Road, Bellville, Cape
Town 7535, South Africa 3135280@myuwc.ac.za; yannik.k.schneider@uit.no;
espen.hansen@uit.no; jeanette.h.andersen@uit.no; johan.isaksson@uit.no;
tbusche@iit-biotech.de; christian.rueckert@iit-biotech.de; joern.kalinowski@iit-
biotech.de; lvanzyl@uwc.ac.za; ituffin@uwc.ac.za 10.3390/md21010002 2022
21 1 - - 2 -
Maloney, Jenny; Molokin, Aleksey; Seguí, Raimundo; Maravilla, Pablo; Martínez-
Hernández, Fernando; Villalobos, Guiehdani; Tsaousis, Anastasios; Gentekaki, Eleni;
Muñoz-Antolí, Carla; Klisiowicz, Debora; Oishi, Camila; Toledo, Rafael; Esteban,
J.; Köster, Pamela; de Lucio, Aida; Dashti, Alejandro; Bailo, Begoña; Calero-
Bernal, Rafael; González-Barrio, David; Carmena, David; Santín, Mónica
Identification and Molecular Characterization of Four New Blastocystis
Subtypes Designated ST35-ST38 Microorganisms EN Article Blastocystis; B.
lapemi; subtype; Oxford Nanopore MinION; ssu rRNA Three recent studies of
Blastocystis epidemiology in mammalian hosts identified four novel sequences that
appeared to share B. lapemi as the most similar sequence. However, full-length ssu
rRNA gene sequences were not available to confirm the validity of these new
subtypes. In the present study, Nanopore MinION sequencing was used to obtain full-
length reference sequences for each of the new subtypes. Additionally, phylogenetic
analyses and pairwise distance comparisons were performed to confirm the validity
of each of these new subtypes. We propose that the novel sequences described in
this study should be assigned the subtype designations ST35-ST38. The full-length
reference sequences of ST35-ST38 will assist in accurate sequence descriptions in
future studies of Blastocystis epidemiology and subtype diversity.
Environmental Microbial and Food Safety Laboratory, Agricultural Research
Service, United States Department of Agriculture, Beltsville, MD 20705, USA
jenny.maloney@usda.gov; aleksey.molokin@usda.gov; raimundo.segui@uv.es;
maravillap@yahoo.com; fherxyz@yahoo.com; guiehda@yahoo.com.mx;
a.tsaousis@kent.ac.uk; gentekaki.ele@mfu.ac.th; carla.munoz@uv.es; deborak@ufpr.br;
camioishi@gmail.com; rafael.toledo@uv.es; jguillermo.esteban@uv.es;
pamelakster@yahoo.com; aida@isciii.es; dashti.alejandro@gmail.com;
begobb@isciii.es; r.calero@ucm.es; dgonzalezbarrio@gmail.com; dacarmena@isciii.es;
monica.santin-duran@usda.gov 10.3390/microorganisms11010046 2022 11 1
- - 46 -
Morales-Rivera, María; Valenzuela-Miranda, Diego; Nuñez-Acuña, Gustavo; Benavente,
Bárbara; Gallardo-Escárate, Cristian; Valenzuela-Muñoz, Valentina Atlantic
Salmon (Salmo salar) Transfer to Seawater by Gradual Salinity Changes Exhibited an
Increase in The Intestinal Microbial Abundance and RichnessMicroorganisms EN
Article Atlantic salmon; parr-smolt transformation; seawater transfer;
intestinal microbiota; nanopore sequencing The host’s physiological
history and environment determine the microbiome structure. In that sense, the
strategy used for the salmon transfer to seawater after parr-smolt transformation
may influence the Atlantic salmon’s intestinal microbiota. Therefore, this
study aimed to explore the diversity and abundance of the Atlantic salmon
intestinal microbiota and metagenome functional prediction during seawater transfer
under three treatments. One group was exposed to gradual salinity change (GSC), the
other to salinity shock (SS), and the third was fed with a functional diet (FD)
before the seawater (SW) transfer. The microbial profile was assessed through full-
16S rRNA gene sequencing using the Nanopore platform. In addition, metagenome
functional prediction was performed using PICRUSt2. The results showed an influence
of salinity changes on Atlantic salmon gut microbiota richness, diversity, and
taxonomic composition. The findings reveal that GSC and the FD increased the
Atlantic salmon smolt microbiota diversity, suggesting a positive association
between the intestinal microbial community and fish health during seawater
transfer. The reported knowledge can be applied to surveil the microbiome in smolt
fish production, improving the performance of Atlantic salmon to seawater transfer.
Interdisciplinary Center for Aquaculture Research (INCAR), University of
Concepción, Concepcion 4030000, Chile marimoralesr@udec.cl;
divalenzuela@udec.cl; gustavonunez@udec.cl; bbenavente@udec.cl;
crisgallardo@oceanografia.udec.cl; valevalenzuela@udec.cl
10.3390/microorganisms11010076 2022 11 1 - - 76 -
Zhang, Tong; Xing, Weiqing; Wang, Aoming; Zhang, Na; Jia, Ling; Ma, Sanyuan; Xia,
Qingyou Comparison of Long-Read Methods for Sequencing and Assembly of
Lepidopteran Pest Genomes International Journal of Molecular Sciences EN
Article biological control; de novo assembly; long-read sequencing;
benchmarking; lepidopteran pest; assembly evaluation Lepidopteran species are
mostly pests, causing serious annual economic losses. High-quality genome
sequencing and assembly uncover the genetic foundation of pest occurrence and
provide guidance for pest control measures. Long-read sequencing technology and
assembly algorithm advances have improved the ability to timeously produce high-
quality genomes. Lepidoptera includes a wide variety of insects with high genetic
diversity and heterozygosity. Therefore, the selection of an appropriate sequencing
and assembly strategy to obtain high-quality genomic information is urgently
needed. This research used silkworm as a model to test genome sequencing and
assembly through high-coverage datasets by de novo assemblies. We report the first
nearly complete telomere-to-telomere reference genome of silkworm Bombyx mori (P50T
strain) produced by Pacific Biosciences (PacBio) HiFi sequencing, and highly
contiguous and complete genome assemblies of two other silkworm strains by Oxford
Nanopore Technologies (ONT) or PacBio continuous long-reads (CLR) that were
unrepresented in the public database. Assembly quality was evaluated by use of
BUSCO, Inspector, and EagleC. It is necessary to choose an appropriate assembler
for draft genome construction, especially for low-depth datasets. For PacBio CLR
and ONT sequencing, NextDenovo is superior. For PacBio HiFi sequencing, hifiasm is
better. Quality assessment is essential for genome assembly and can provide better
and more accurate results. For chromosome-level high-quality genome construction,
we recommend using 3D-DNA with EagleC evaluation. Our study references how to
obtain and evaluate high-quality genome assemblies, and is a resource for
biological control, comparative genomics, and evolutionary studies of Lepidopteran
pests and related species. State Key Laboratory of Silkworm Genome Biology,
Biological Science Research Center, Southwest University, Chongqing 400715, China
zt137703197@email.swu.edu.cn; xingwq@email.swu.edu.cn; 18838934811@163.com;
zhangna1522@outlook.com; jialing132@163.com; masy@swu.edu.cn; xiaqy@swu.edu.cn
10.3390/ijms24010649 2022 24 1 - - 649 -
Abdulaziz, Anas; Pramodh, Athira; Sukumaran, Vrinda; Raj, Devika; John, Ann The
Influence of Photodynamic Antimicrobial Chemotherapy on the Microbiome,
Neuroendocrine and Immune System of Crustacean Post Larvae Toxics EN
Article photodynamic therapy; curcumin; water-associated pathogen;
reactive oxygen; aquaculture; neuroendocrine toxicity Photodynamic antimicrobial
chemotherapy (PACT), employing a combination of light and natural photosensitizer
molecules such as curcumin, has been accepted as a safe modality for removing
aquatic pathogens which cause diseases such as cholera in humans and vibriosis in
aquatic animals. Curcumin and its photodegradation products are generally
considered as safe to animals, but the impact of reactive oxygen species (ROS)
generated by these products on the growth and survival of organisms at a cellular
level has not been studied in detail. The ROS generated by curcumin on
photoexcitation using blue light (λmax 405 nm, 10 mW cm−2) disinfects
more than 80% of free-living Vibrio spp. in the rearing water of Penaeus monodon.
However, it is less effective against Vibrio spp. colonized inside P. monodon
because the carapace of the animal prevents the transmission of more than 70% of
light at the 400–450 nm range and thus reduces the formation of ROS. The
influence of curcumin and photoexcited curcumin on the microbiome of P. monodon
were revealed by nanopore sequencing. The photoexcited curcumin induced irregular
expression of genes coding the moult-inhibiting hormone (MIH), Crustacean
hyperglycaemic hormone (CHH)), prophenoloxidase (ProPO), and crustin, which
indicates toxic effects of ROS generated by photoexcited curcumin on the
neuroendocrine and immune systems of crustaceans, which could alter their growth
and survival in aquaculture settings. The study proposed the cautious use of
photodynamic therapy in aquaculture systems, and care must be taken to avoid
photoexcitation when animals are experiencing moulting or environmental stress.
CSIR-National Institute of Oceanography, Regional Centre Kochi, Cochin
682018, Kerala, India anas@nio.org; athira06041998@gmail.com;
vrindas@cusat.ac.in; rajkdevika@gmail.com; annmary.11214@gmail.com
10.3390/toxics11010036 2022 11 1 - - 36 -
Buttler, Jeremy; Drown, Devin Accuracy and Completeness of Long Read Metagenomic
Assemblies Microorganisms EN Article nanopore sequencing; benchmarking;
microbial communities; long read assemblers Microbes influence the surrounding
environment and contribute to human health. Metagenomics can be used as a tool to
explore the interactions between microbes. Metagenomic assemblies built using long
read nanopore data depend on the read level accuracy. The read level accuracy of
nanopore sequencing has made dramatic improvements over the past several years.
However, we do not know if the increased read level accuracy allows for faster
assemblers to make as accurate metagenomic assemblies as slower assemblers. Here,
we present the results of a benchmarking study comparing three commonly used long
read assemblers, Flye, Raven, and Redbean. We used a prepared DNA standard of seven
bacteria as our input community. We prepared a sequencing library using a VolTRAX
V2 and sequenced using a MinION mk1b. We basecalled with Guppy v5.0.7 using the
super-accuracy model. We found that increasing read depth benefited each of the
assemblers, and nearly complete community member chromosomes were assembled with as
little as 10× read depth. Polishing assemblies using Medaka had a predictable
improvement in quality. We found Flye to be the most robust across taxa and was the
most effective assembler for recovering plasmids. Based on Flye’s consistency
for chromosomes and increased effectiveness at assembling plasmids, we would
recommend using Flye in future metagenomic studies. Department of Biology and
Wildlife, University of Alaska Fairbanks, Fairbanks, AK 99775, USA
jdbuttler@alaska.edu; dmdrown@alaska.edu 10.3390/microorganisms11010096
2022 11 1 - - 96 -
Romero-Calle, Danitza; Pedrosa-Silva, Francisnei; Tomé, Luiz; Sousa, Thiago; de
Oliveira Santos, Leila; de Carvalho Azevedo, Vasco; Brenig, Bertram; Benevides,
Raquel; Venancio, Thiago; Billington, Craig; Góes-Neto, Aristóteles Hybrid
Genomic Analysis of Salmonella enterica Serovar Enteritidis SE3 Isolated from
Polluted Soil in Brazil Microorganisms EN Article whole genome sequencing;
Salmonella; hybrid sequence assembly; heavy metal; antimicrobial resistance In
Brazil, Salmonella enterica serovar Enteritidis is a significant health threat.
Salmonella enterica serovar Enteritidis SE3 was isolated from soil at the
Subaé River in Santo Amaro, Brazil, a region contaminated with heavy metals
and organic waste. Illumina HiSeq and Oxford Nanopore Technologies MinION
sequencing were used for de novo hybrid assembly of the Salmonella SE3 genome. This
approach yielded 10 contigs with 99.98% identity with S. enterica serovar
Enteritidis OLF-SE2-98984-6. Twelve Salmonella pathogenic islands, multiple
virulence genes, multiple antimicrobial gene resistance genes, seven phage defense
systems, seven prophages and a heavy metal resistance gene were encoded in the
genome. Pangenome analysis of the S. enterica clade, including Salmonella SE3,
revealed an open pangenome, with a core genome of 2137 genes. Our study showed the
effectiveness of a hybrid sequence assembly approach for environmental Salmonella
genome analysis using HiSeq and MinION data. This approach enabled the
identification of key resistance and virulence genes, and these data are important
to inform the control of Salmonella and heavy metal pollution in the Santo Amaro
region of Brazil. Postgraduate Program in Biotechnology, State University of Feira
de Santana (UEFS), Av. Transnordestina S/N, Feira de Santana 44036-900, BA, Brazil
ppgbiotec@uefs.br; francisneipedrosa@gmail.com; marcelofsa_rt@hotmail.com;
thiagojsousa@gmail.com; leilathaise@yahoo.com.br; vascoariston@gmail.com;
bbrenig@gwdg.de; raquelgb@gmail.com; thiago.venancio@gmail.com;
craig.billington@esr.cri.nz; arigoesneto@icb.ufmg.br
10.3390/microorganisms11010111 2022 11 1 - - 111 -
Lopatriello, Giulia; Maestri, Simone; Alfano, Massimiliano; Papa, Roberto; Di
Vittori, Valerio; De Antoni, Luca; Bellucci, Elisa; Pieri, Alice; Bitocchi, Elena;
Delledonne, Massimo; Rossato, Marzia CRISPR/Cas9-Mediated Enrichment Coupled
to Nanopore Sequencing Provides a Valuable Tool for the Precise Reconstruction of
Large Genomic Target Regions International Journal of Molecular Sciences EN
Article de novo assembly; variant calling; Cas9-tiling enrichment;
nanopore sequencing; pod-shattering Complete and accurate identification of genetic
variants associated with specific phenotypes can be challenging when there is a
high level of genomic divergence between individuals in a study and the
corresponding reference genome. We have applied the Cas9-mediated enrichment
coupled to nanopore sequencing to perform a targeted de novo assembly and
accurately reconstruct a genomic region of interest. This approach was used to
reconstruct a 250-kbp target region on chromosome 5 of the common bean genome
(Phaseolus vulgaris) associated with the shattering phenotype. Comparing a non-
shattering cultivar (Midas) with the reference genome revealed many single-
nucleotide variants and structural variants in this region. We cut five 50-kbp
tiled sub-regions of Midas genomic DNA using Cas9, followed by sequencing on a
MinION device and de novo assembly, generating a single contig spanning the whole
250-kbp region. This assembly increased the number of Illumina reads mapping to
genes in the region, improving their genotypability for downstream analysis. The
Cas9 tiling approach for target enrichment and sequencing is a valuable alternative
to whole-genome sequencing for the assembly of ultra-long regions of interest,
improving the accuracy of downstream genotype–phenotype association analysis.
Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134
Verona, Italy giulia.lopatriello@univr.it; simone.maestri@univr.it;
massimiliano.alfano@univr.it; r.papa@staff.univpm.it; v.divittori@staff.univpm.it;
luca.deantoni@univr.it; e.bellucci@staff.univpm.it; a.pieri@staff.univpm.it;
e.bitocchi@staff.univpm.it; massimo.delledonne@univr.it; marzia.rossato@univr.it
10.3390/ijms24021076 2023 24 2 - - 1076 -
Nowlan, Joseph; Sies, Ashton; Britney, Scott; Cameron, Andrew; Siah, Ahmed;
Lumsden, John; Russell, Spencer Genomics of Tenacibaculum Species in British
Columbia, Canada Pathogens EN Article phylogenetics; de novo assembly;
virulence; antimicrobial resistance; diversity; Tenacibaculum; mouthrot; Atlantic
salmon Tenacibaculum is a genus of Gram-negative filamentous bacteria with a
cosmopolitan distribution. The research describing Tenacibaculum genomes stems
primarily from Norway and Chile due to their impacts on salmon aquaculture.
Canadian salmon aquaculture also experiences mortality events related to the
presence of Tenacibaculum spp., yet no Canadian Tenacibaculum genomes are publicly
available. Ribosomal DNA sequencing of 16S and four species-specific 16S
quantitative-PCR assays were used to select isolates cultured from Atlantic salmon
with mouthrot in British Columbia (BC), Canada. Ten isolates representing four
known and two unknown species of Tenacibaculum were selected for shotgun whole
genome sequencing using the Oxford Nanopore’s MinION platform. The genome
assemblies achieved closed circular chromosomes for seven isolates and long contigs
for the remaining three isolates. Average nucleotide identity analysis identified
T. ovolyticum, T. maritimum, T. dicentrarchi, two genomovars of T. finnmarkense,
and two proposed novel species T. pacificus sp. nov. type strain 18-2881-AT and T.
retecalamus sp. nov. type strain 18-3228-7BT. Annotation in most of the isolates
predicted putative virulence and antimicrobial resistance genes, most-notably
toxins (i.e., hemolysins), type-IX secretion systems, and oxytetracycline
resistance. Comparative analysis with the T. maritimum type-strain predicted
additional toxins and numerous C-terminal secretion proteins, including an M12B
family metalloprotease in the T. maritimum isolates from BC. The genomic prediction
of virulence-associated genes provides important targets for studies of mouthrot
disease, and the annotation of the antimicrobial resistance genes provides targets
for surveillance and diagnosis in veterinary medicine. Center for Innovation in
Fish Health, Vancouver Island University, Nanaimo, BC V9R 5S5, Canada
joseph.nowlan@viu.ca; ans227@uregina.ca; scott.britney@viu.ca;
andrew.cameron@uregina.ca; ahmed.siah@cahs-bc.ca; jsl@ovc.uoguelph.ca;
spencer.russell@viu.ca 10.3390/pathogens12010101 2023 12 1 - -
101 -
Lesbayev, Bakhytzhan; Auyelkhankyzy, Moldir; Ustayeva, Gaukhar; Yeleuov, Mukhtar;
Rakhymzhan, Nurgali; Maral, Yerkebulan; Tolynbekov, Aidos Modification of Biomass-
Derived Nanoporous Carbon with Nickel Oxide Nanoparticles for Supercapacitor
Application Journal of Composites Science EN Article biomass-derived; walnut
shell; activated carbon; modification; CVD; supercapacitorsSupercapacitors are one
of the promising devices for the accumulation and storage of electrical energy. The
purpose of this study is to develop a synthesis and modification method of carbon
material to improve the electrochemical characteristics of a supercapacitor. In the
proposed study, by varying the sequence and parameters of the processes of
carbonization, mechanoactivation and thermochemical activation, the conditions for
obtaining nanoporous carbon with a specific surface area of 2200 (±50) m2/g
from walnut shells (WSs) are optimized. In addition, to increase the
electrochemical efficiency of the electrode material, the resulting nanoporous
carbon was modified with nickel oxide (NiO) nanoparticles by the thermochemical
method. It is shown that the modification with nickel oxide nanoparticles makes it
possible to increase the specific capacitance of the supercapacitor electrode by
16% compared to the original unmodified nanoporous carbon material. Faculty of
Chemistry and Chemical Technology, al-Farabi Kazakh National University, Almaty
050040, Kazakhstan bakytzhan.lesbayev@kaznu.edu.kz;
moldir.auyelkhankyzy@kaznu.edu.kz; gaukhar.sakenovna@gmail.com;
mukhtar.yu@gmail.com; nurrts@mail.ru; maralerkebulan07@gmail.com;
a.tolynbekov@gmail.com 10.3390/jcs7010020 2023 7 1 - - 20
-
Baehren, Carolin; Pembaur, Anton; Weil, Patrick; Wewers, Nora; Schult, Frank;
Wirth, Stefan; Postberg, Jan; Aydin, Malik The Overlooked
Microbiome—Considering Archaea and Eukaryotes Using Multiplex
Nanopore-16S-/18S-rDNA-Sequencing: A Technical Report Focusing on Nasopharyngeal
Microbiomes International Journal of Molecular Sciences EN Article
archaeome; archaea; eukaryotes; PCR; sequencing; MinION; respiratory diseases
In contrast to bacteria, microbiome analyses often neglect archaea, but also
eukaryotes. This is partly because they are difficult to culture due to their
demanding growth requirements, or some even have to be classified as uncultured
microorganisms. Consequently, little is known about the relevance of archaea in
human health and diseases. Contemporary broad availability and spread of next
generation sequencing techniques now enable a stronger focus on such
microorganisms, whose cultivation is difficult. However, due to the enormous
evolutionary distances between bacteria, archaea and eukaryotes, the implementation
of sequencing strategies for smaller laboratory scales needs to be refined to
achieve as a holistic view on the microbiome as possible. Here, we present a
technical approach that enables simultaneous analyses of archaeal, bacterial and
eukaryotic microbial communities to study their roles in development and courses of
respiratory disorders. We thus applied combinatorial 16S-/18S-rDNA sequencing
strategies for sequencing-library preparation. Considering the lower total
microbiota density of airway surfaces, when compared with gut microbiota, we
optimized the DNA purification workflow from nasopharyngeal swab specimens. As a
result, we provide a protocol that allows the efficient combination of bacterial,
archaeal, and eukaryotic libraries for nanopore-sequencing using Oxford Nanopore
Technologies MinION devices and subsequent phylogenetic analyses. In a pilot study,
this workflow allowed the identification of some environmental archaea, which were
not correlated with airway microbial communities before. Moreover, we assessed the
protocol’s broader applicability using a set of human stool samples. We
conclude that the proposed protocol provides a versatile and adaptable tool for
combinatorial studies on bacterial, archaeal, and eukaryotic microbiomes on a small
laboratory scale. Laboratory of Experimental Pediatric Pneumology and Allergology,
Center for Biomedical Education and Research, School of Life Sciences (ZBAF),
Faculty of Health, Witten/Herdecke University, 58455 Witten, Germany
carolin.baehren@uni-wh.de; anton.pembaur@uni-wh.de; patrick.weil@uni-wh.de;
nora.wewers@uni-wh.de; frank.schult@uni-due.de; stefan.wirth@uni-wh.de;
jan.postberg@uni-wh.de; malik.aydin@uni-wh.de 10.3390/ijms24021426 2023 24
2 - - 1426 -
Tamayo, Alejandra; Núñez-Moreno, Gonzalo; Ruiz, Carolina; Plaisancie, Julie;
Damian, Alejandra; Moya, Jennifer; Chassaing, Nicolas; Calvas, Patrick; Ayuso,
Carmen; Minguez, Pablo; Corton, Marta Minigene Splicing Assays and Long-Read
Sequencing to Unravel Pathogenic Deep-Intronic Variants in PAX6 in Congenital
Aniridia International Journal of Molecular Sciences EN Article PAX6;
congenital aniridia; non-canonical splicing sites; deep-intronic variants; minigene
splicing assays; long-read sequencing; MinION nanopore sequencing PAX6
haploinsufficiency causes aniridia, a congenital eye disorder that involves the
iris, and foveal hypoplasia. Comprehensive screening of the PAX6 locus, including
the non-coding regions, by next-generation sequencing revealed four deep-intronic
variants with potential effects on pre-RNA splicing. Nevertheless, without a
functional analysis, their pathogenicity could not be established. We aimed to
decipher their impact on the canonical PAX6 splicing using in vitro minigene
splicing assays and nanopore-based long-read sequencing. Two multi-exonic PAX6
constructs were generated, and minigene assays were carried out. An aberrant
splicing pattern was observed for two variants in intron 6, c.357+136G>A and
c.357+334G>A. In both cases, several exonization events, such as pseudoexon
inclusions and partial intronic retention, were observed due to the creation or
activation of new/cryptic non-canonical splicing sites, including a shared intronic
donor site. In contrast, two variants identified in intron 11, c.1032+170A>T and
c.1033-275A>C, seemed not to affect splicing processes. We confirmed the high
complexity of alternative splicing of PAX6 exon 6, which also involves unreported
cryptic intronic sites. Our study highlights the importance of integrating
functional studies into diagnostic algorithms to decipher the potential implication
of non-coding variants, usually classified as variants of unknown significance,
thus allowing variant reclassification to achieve a conclusive genetic diagnosis.
Department of Genetics & Genomics, Instituto de Investigación Sanitaria-
Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-
FJD, UAM), 28040 Madrid, Spain alejandratamayoduran@gmail.com;
gonzalo.nunezm@quironsalud.es; carolina.ruchez13@gmail.com; plaisancie.j@chu-
toulouse.fr; alejandra.damian@quironsalud.es; jmoyavaquero@gmail.com;
chassaing.n@chu-toulouse.fr; calvas.p@chu-toulouse.fr; cayuso@fjd.es;
pablo.minguez@quironsalud.es; mcorton@fjd.es 10.3390/ijms24021562 2023 24
2 - - 1562 -
Jeong, Chanyoung; Jung, Jeki; Sheppard, Keith; Choi, Chang-Hwan Control of the
Nanopore Architecture of Anodic Alumina via Stepwise Anodization with Voltage
Modulation and Pore Widening Nanomaterials EN Article nanopore; alumina;
anodization; hierarchical nanostructures; hybrid nanostructures Control of the
morphology and hierarchy of the nanopore structures of anodic alumina is
investigated by employing stepwise anodizing processes, alternating the two
different anodizing modes, including mild anodization (MA) and hard anodization
(HA), which are further mediated by a pore-widening (PW) step in between. For the
experiment, the MA and HA are applied at the anodizing voltages of 40 and 100 V,
respectively, in 0.3 M oxalic acid, at 1 °C, for fixed durations (30 min for MA
and 0.5 min for HA), while the intermediate PW is applied in 0.1 M phosphoric acid
at 30 °C for different durations. In particular, to examine the effects of the
anodizing sequence and the PW time on the morphology and hierarchy of the nanopore
structures formed, the stepwise anodization is conducted in two different ways: one
with no PW step, such as MA→HA and HA→MA, and the other with the timed PW
in between, such as MA→PW→MA, MA→PW→HA, HA→PW→HA, and
HA→PW→MA. The results show that both the sequence of the voltage-
modulated anodizing modes and the application of the intermediate PW step led to
unique three-dimensional morphology and hierarchy of the nanopore structures of the
anodic alumina beyond the conventional two-dimensional cylindrical pore geometry.
It suggests that the stepwise anodizing process regulated by the sequence of the
anodizing modes and the intermediate PW step can allow the design and fabrication
of various types of nanopore structures, which can broaden the applications of the
nanoporous anodic alumina with greater efficacy and versatility. Department of
Advanced Materials Engineering, Dong-eui University, Busan 47340, Republic of Korea
cjeong@deu.ac.kr; jjung8@stevens.edu; keith.sheppard@stevens.edu;
cchoi@stevens.edu 10.3390/nano13020342 2023 13 2 - - 342 -
Adrian, M; Poerwanto, Roedhy; Inoue, Eiichi; Matra, Deden Transcriptome Dataset of
Strawberry (Fragaria × ananassa Duch.) Leaves Using Oxford Nanopore
Sequencing under LED Irradiation and Application of Methyl Jasmonate and Methyl
Salicylate Hormones Treatment Data EN Data Descriptor strawberry; long-read
sequencing; lights; plant hormone; RNASeq This data descriptor introduces a
transcriptome dataset of strawberry plant left exposed to an LED light treatment
and plant hormones of Methyl Jasmonate (MeJA) and Methyl Salicylate (MeSA). These
data consist of a transcriptome dataset (four libraries) obtained from the leaves
of strawberry plants treated with LEDs of blue and red spectrums and the hormones
of Methyl Jasmonate (MeJA) and Methyl Salicylate (MeSA), which allowed us to
conduct a further analysis of the growth and development processes of strawberry
plants. In addition, we describe detailed procedures on how the plants were
prepared and treated and how the data were generated and processed beforehand.
Further analysis of these data will significantly help to improve our understanding
of the molecular mechanisms of LED light and MeJA-MeSA in strawberry plants.
Agronomy and Horticulture Study Program, Graduate School, IPB University,
Dramaga Bogor 16680, Indonesia mehmedadrian@apps.ipb.ac.id;
roedhy@apps.ipb.ac.id; eiichi.inoue.a@vc.ibaraki.ac.jp; dedenmatra@apps.ipb.ac.id
10.3390/data8020022 2023 8 2 - - 22 -
Rocchigiani, Angela; Ferretti, Luca; Ledda, Alice; Di Nardo, Antonello; Floris,
Matteo; Bonelli, Piero; Loi, Federica; Idda, Maria; Angioi, Pier; Zinellu, Susanna;
Fiori, Mariangela; Bechere, Roberto; Capitta, Paola; Coccollone, Annamaria;
Coradduzza, Elisabetta; Dettori, Maria; Fattaccio, Maria; Gallisai, Elena;
Maestrale, Caterina; Manunta, Daniela; Pedditzi, Aureliana; Piredda, Ivana; Palmas,
Bruna; Salza, Sara; Sechi, Anna; Tanda, Barbara; Madrau, Maria; Sanna, Maria;
Cherchi, Simonetta; Ponti, Nicoletta; Masala, Giovanna; Sirica, Roberto;
Evangelista, Eloisa; Oggiano, Annalisa; Puggioni, Giantonella; Ligios, Ciriaco; Dei
Giudici, Silvia Origin, Genetic Variation and Molecular Epidemiology of SARS-CoV-
2 Strains Circulating in Sardinia (Italy) during the First and Second COVID-19
Epidemic Waves Viruses EN Article SARS-CoV-2; spike (S) protein;
whole genome sequencing; molecular epidemiology; genetic diversity
Understanding how geography and human mobility shape the patterns and spread
of infectious diseases such as COVID-19 is key to control future epidemics. An
interesting example is provided by the second wave of the COVID-19 epidemic in
Europe, which was facilitated by the intense movement of tourists around the
Mediterranean coast in summer 2020. The Italian island of Sardinia is a major
tourist destination and is widely believed to be the origin of the second Italian
wave. In this study, we characterize the genetic variation among SARS-CoV-2 strains
circulating in northern Sardinia during the first and second Italian waves using
both Illumina and Oxford Nanopore Technologies Next Generation Sequencing methods.
Most viruses were placed into a single clade, implying that despite substantial
virus inflow, most outbreaks did not spread widely. The second epidemic wave on the
island was actually driven by local transmission of a single B.1.177 subclade.
Phylogeographic analyses further suggest that those viral strains circulating on
the island were not a relevant source for the second epidemic wave in Italy. This
result, however, does not rule out the possibility of intense mixing and
transmission of the virus among tourists as a major contributor to the second
Italian wave. Istituto Zooprofilattico Sperimentale della Sardegna, 07100
Sassari, Italy angelamaria.rocchigiani@izs-sardegna.it; luca.ferretti@gmail.com;
alice.ledda@ukhsa.gov.uk; antonello.di-nardo@pirbright.ac.uk;
matteo.floris@gmail.com; piero.bonelli@izs-sardegna.it; federica.loi@izs-
sardegna.it; m.laurai@yahoo.it; pierpaolo.angioi@izs-sardegna.it;
susanna.zinellu@izs-sardegna.it; mariangela.fiori@izs-sardegna.it;
roberto.bechere@izs-sardegna.it; paola.capitta@izs-sardegna.it;
annamaria.coccollone@izs-sardegna.it; elisabetta.coradduzza@izs-sardegna.it;
mariaantoniettadettori@izs-sardegna.it; caterina.fattaccio@izs-sardegna.it;
elena.gallisai@izs-sardegna.it; caterina.maestrale@izs-sardegna.it;
daniela.manunta@izs-sardegna.it; aureliana.pedditzi@izs-sardegna.it;
ivana.piredda@izs-sardegna.it; bruna.palmas@izs-sardegna.it; sara.salza@izs-
sardegna.it; annamaria.sechi@izs-sardegna.it; barbara.tanda@izs-sardegna.it;
paola.madrau@izs-sardegna.it; marialuisa.sanna@izs-sardegna.it;
simonetta.cherchi@izs-sardegna.it; marianicoletta.ponti@gmail.com;
giovanna.masala@izs-sardegna.it; roberto.sirica@centroames.it;
elo.evangelista@gmail.com; annalisa.oggiano@izs-sardegna.it;
giantonella.puggioni@izs-sardegna.it; ciriaco.ligios@izs-sardegna.it;
silvia.deigiudici@izs-sardegna.it 10.3390/v15020277 2023 15 2 - -
277 -
Polkhovskaya, Ekaterina; Bolotina, Anna; Merkulov, Pavel; Dudnikov, Maxim;
Soloviev, Alexander; Kirov, Ilya Long-Read cDNA Sequencing Revealed Novel
Expressed Genes and Dynamic Transcriptome Landscape of Triticale (x Triticosecale
Wittmack) Seed at Different Developing Stages Agronomy EN Communication
Nanopore sequencing; transcriptome; triticale; seed development; annotation
 Developing seed is a unique stage of plant development with highly dynamic
changes in transcriptome. Here, we aimed to detect the novel previously
unannotated, genes of the triticale (x Triticosecale Wittmack, AABBRR genome
constitution) genome that are expressed during different stages and at different
parts of the developing seed. For this, we carried out the Oxford Nanopore
sequencing of cDNA obtained for middle (15 days post-anthesis, dpa) and late (20
dpa) stages of seed development. The obtained data together with our previous
direct RNA sequencing of early stage (10 dpa) of seed development revealed 39,914
expressed genes including 7128 (17.6%) genes that were not previously annotated in
A, B, and R genomes. The bioinformatic analysis showed that the identified genes
belonged to long non-coding RNAs (lncRNAs), protein-coding RNAs, and TE-derived
RNAs. The gene set analysis revealed the transcriptome dynamics during seed
development with distinct patterns of over-represented gene functions in early and
middle/late stages. We performed analysis of the lncRNA genes polymorphism and
showed that the genes of some of the tested lncRNAs are indeed polymorphic in the
triticale collection. Altogether, our results provide information on thousands of
novel loci expressed during seed development that can be used as new targets for
GWAS analysis, the marker-assisted breeding of triticale, and functional
elucidation. All-Russia Research Institute of Agricultural Biotechnology,
Moscow 127550, Russia eynzeynkreyn@gmail.com; boloti.anya@yandex.ru;
paulmerkulov97@gmail.com; max.dudnikov.07@gmail.com; a.soloviev70@gmail.com;
kirovez@gmail.com 10.3390/agronomy13020292 2023 13 2 - - 292
-
Luo, Lan; Fu, Aisi; Shi, Manman; Hu, Jiawei; Kong, Deguang; Liu, Tiangang; Yuan,
Jingping; Sun, Shengrong; Chen, Chuang Species-Level Characterization of the
Microbiome in Breast Tissues with Different Malignancy and Hormone-Receptor
Statuses Using Nanopore Sequencing Journal of Personalized Medicine EN
Article microbiome; breast cancer; 16S rRNA; nanopore sequencing;
diversity Unambiguous evidence indicates that microbes are closely linked to
various human diseases, including cancer. Most prior work investigating the
microbiome of breast tissue describes an association between compositional
differences of microbial species in benign and malignant tissues, but few studies
have examined the relative abundance of microbial communities within human breast
tissue at the species level. In this work, a total of 44 breast tissue samples
including benign and malignant tissues with adjacent normal breast tissue pairs
were collected, and Oxford Nanopore long-read sequencing was employed to assess
breast tissue microbial signatures. Nearly 900 bacterial species were detected from
the four dominant phyla: Proteobacteria, Firmicutes, Actinobacteria and
Bacteroidetes. The bacteria with the highest abundance in all breast tissues was
Ralstonia pickettii, and its relative abundance increased with decreasing
malignancy. We further examined the breast-tissue microbiome composition with
different hormone-receptor statuses, and the relative abundance of the genus
Pseudomonas increased most significantly in breast tissues. Our study provides a
rationale for exploring microbiomes associated with breast carcinogenesis and
cancer development. Further large-cohort investigation of the breast microbiome is
necessary to characterize a microbial risk signature and develop potential
microbial-based prevention therapies. Department of Thyroid and Breast Surgery,
Renmin Hospital of Wuhan University, Wuhan 430060, China lanluo@whu.edu.cn;
fuaisi@dgensee.com; mammans@126.com; hujiawei@whu.edu.cn; kongdeguang08@126.com;
liutg@whu.edu.cn; yuanjingping2003@aliyun.com; sun137@sina.com; chenc0678@126.com
10.3390/jpm13020174 2023 13 2 - - 174 -
García-Campa, Lara; Valledor, Luis; Pascual, Jesús The Integration of Data from
Different Long-Read Sequencing Platforms Enhances Proteoform Characterization in
Arabidopsis Plants EN Article proteogenomics; long-read; sequencing;
nanopore; PacBio; protein database; proteoform; ONT-DRS; Iso-Seq The increasing
availability of massive omics data requires improving the quality of reference
databases and their annotations. The combination of full-length isoform sequencing
(Iso-Seq) with short-read transcriptomics and proteomics has been successfully used
for increasing proteoform characterization, which is a main ongoing goal in
biology. However, the potential of including Oxford Nanopore Technologies Direct
RNA Sequencing (ONT-DRS) data has not been explored. In this paper, we analyzed the
impact of combining Iso-Seq- and ONT-DRS-derived data on the identification of
proteoforms in Arabidopsis MS proteomics data. To this end, we selected a
proteomics dataset corresponding to senescent leaves and we performed protein
searches using three different protein databases: AtRTD2 and AtRTD3, built from the
homonymous transcriptomes, regarded as the most complete and up-to-date available
for the species; and a custom hybrid database combining AtRTD3 with publicly
available ONT-DRS transcriptomics data generated from Arabidopsis leaves. Our
results show that the inclusion and combination of long-read sequencing data from
Iso-Seq and ONT-DRS into a proteogenomic workflow enhances proteoform
characterization and discovery in bottom-up proteomics studies. This represents a
great opportunity to further investigate biological systems at an unprecedented
scale, although it brings challenges to current protein searching algorithms.
Plant Physiology, Department of Organisms and Systems Biology, University of
Oviedo, 33003 Oviedo, Spain garciaclara@uniovi.es; valledorluis@uniovi.es;
pascualjesus@uniovi.es 10.3390/plants12030511 2023 12 3 - - 511
-
Kosewska, Olga; Przemieniecki, Sebastian; Nietupski, Mariusz The Effect of
Antibiotics on Bacteriome of Sitophilus oryzae and Rhyzopertha dominica as a Factor
Determining the Success of Foraging: A Chance for Antibiotic Therapy in Grain
Stores Applied Sciences EN Article symbiotic microbiome; storage
pests; nanopore sequencing; biochemical activities Rice weevil (Sitophilus
oryzae) and the lesser grain borer (Rhyzopertha dominica) are very important
warehouse pests and, therefore, their control is crucial. At a key moment in the
life of adult Sitophilus spp., the obligatory symbiotic nature of the Sodalis
pierantonius bacterium opens up a new perspective for natural antibiotics and
bactericides. In this study, we used nanopore sequencing for 16S rRNA barcoding to
evaluate the internal bacteriome of S. oryzae and R. dominica and sterilized the
insects’ internal microbiome with gentamicin. The treatment of the interior
of S. oryzae with gentamicin (30 mg·g−1) hampered insect functioning
(supposed lack of DOPA (4-dihydroxyphenylalanine) synthesis, stabilizing the
exoskeleton by Sodalis pierantonius symbiont) and elicited a lethal effect in the
first stages of this pest’s adult life. In addition, we identified
biochemical biomarkers (enzymatic activity and substrate utilization) active in
living individuals, but inactive in dead individuals (e.g., C8 esterase/lipase and
α-chymotrypsin). Department of Entomology, Phytopathology and Molecular
Diagnostics, University of Warmia and Mazury in Olsztyn, Prawocheńskiego 17, 10-720
Olsztyn, Poland olga.kosewska@uwm.edu.pl; sebastian.przemieniecki@uwm.edu.pl;
maniek@uwm.edu.pl 10.3390/app13031576 2023 13 3 - - 1576 -
Vasquez, Ignacio; Retamales, Julio; Parra, Barbara; Machimbirike, Vimbai; Robeson,
James; Santander, Javier Comparative Genomics of a Polyvalent Escherichia-
Salmonella Phage fp01 and In Silico Analysis of Its Receptor Binding Protein and
Conserved Enterobacteriaceae Phage Receptor Viruses EN Article
polyvalent bacteriophage fp01; Escherichia coli; Salmonella; genome The
polyvalent bacteriophage fp01, isolated from wastewater in Valparaiso, Chile, was
described to have lytic activity across bacterial species, including Escherichia
coli and Salmonella enterica serovars. Due to its polyvalent nature, the
bacteriophage fp01 has potential applications in the biomedical, food and
agricultural industries. Also, fundamental aspects of polyvalent bacteriophage
biology are unknown. In this study, we sequenced and described the complete genome
of the polyvalent phage fp01 (MH745368.2) using long- (MinION, Nanopore) and short-
reads (MiSeq, Illumina) sequencing. The bacteriophage fp01 genome has 109,515 bp,
double-stranded DNA with an average G+C content of 39%, and 158 coding sequences
(CDSs). Phage fp01 has genes with high similarity to Escherichia coli, Salmonella
enterica, and Shigella sp. phages. Phylogenetic analyses indicated that the phage
fp01 is a new Tequintavirus fp01 specie. Receptor binding protein gp108 was
identified as potentially responsible for fp01 polyvalent characteristics, which
binds to conserved amino acid regions of the FhuA receptor of Enterobacteriaceae.
 Marine Microbial Pathogenesis and Vaccinology Laboratory, Department of Ocean
Science, Memorial University, St. John’s, NL A1C 5S7, Canada
ivasquezsoli@mun.ca; retamales@udla.c; bparra@ispch.cl; imachimbirik@mun.ca;
james.robeson@pucv.cl; jsantander@mun.ca 10.3390/v15020379 2023 15 2 -
- 379 -
Eguchi, Hiroshi; Hotta, Fumika; Kusaka, Shunji Applying Metagenomic Analysis Using
Nanopore Sequencer (MinION) for Precision Medicine in Bacterial
Keratoconjunctivitis: Comprehensive Validation of Molecular Biological and
Conventional Examinations International Journal of Molecular Sciences EN
Article bacterial keratoconjunctivitis; corneal scraping; culture; smear
microscopic examination; nanopore sequencer (MinION) Smear microscopic examination
and culture of the corneal scrapings are the gold standards for the diagnosis of
bacterial keratoconjunctivitis. High-sensitivity molecular biological examinations
of the ocular surface specimens are used clinically. However, the results require
careful interpretation to avoid the unintentional detection of indigenous bacteria.
Results of conventional and state-of-the-art examinations require clinical
verification for specificity and sensitivity. In this study, smear microscopic
examination, culture, and nanopore sequencing using the MinION of ocular surface
specimens from eight clinically diagnosed bacterial keratoconjunctivitis cases were
performed and compared. Seven of the eight cases (87.5%) were smear positive and
five (62.5%) were culture positive. The former showed the same genus in >60% of
the classified reads as one specific bacterium inferred from the smear microscopy
when sequenced by the MinION. In two of the three culture-negative cases, the
smear-positive images were highly reminiscent of the species comprising most of the
MinION sequences. Four of the five culture-positive cases were consistent with the
most prevalent bacteria in the sequencing results. Probable contamination among
specimens processed on the same day were observed. In conclusion, the microscopic
examination of the corneal scraping specimens may be more sensitive and specific
than the culture examination. Additionally, although metagenomic analysis using the
MinION contributes to more precise medication for bacterial keratoconjunctivitis,
contamination can affect the results. Department of Ophthalmology, Kindai
University, Osakasayama 589-8511, Japan hiroegu0113@gmail.com;
uri0428@yahoo.co.jp; skusaka@gmail.com 10.3390/ijms24032611 2023 24 3
- - 2611 -
Stainton, Kirsty; McGreig, Sam; Conyers, Chris; Ponting, Sally; Butler, Lee; Brown,
Paul; Jones, Eleanor Molecular Identification of Asian Hornet Vespa velutina
nigrithorax Prey from Larval Gut Contents: A Promising Method to Study the Diet of
an Invasive Pest Animals EN Article Apis mellifera; apiculture; Vespa
velutina; invasive species; diet metabarcoding The Asian hornet, Vespa velutina
nigrithorax (Hymenoptera: Vespidae), is an invasive hornet that was accidentally
introduced into Europe in 2004. It mainly preys on other invertebrates and
arthropod species, and often targets honey bee (Apis mellifera) colonies. The
introduction of these hornets may damage indigenous fauna and apiculture. Knowledge
of V. velutina prey preference and the species composition of their diet is
relatively limited. In this study, we assessed methodologies for the molecular
identification of prey using dissected larvae from destroyed nests. Ten larval
samples were taken from five nests in areas where the hornets had not yet
established: two from the Channel Islands and three in the mainland UK. DNA was
extracted from the gut contents and sequenced and analysed by metabarcoding with
Oxford Nanopore Technologies’ Flongle and MinION devices. Numerous taxa were
detected in each larval sample with the species composition varying by individual
and by nest. Between 15 and 26 species were found per nest, with wasps (Vespula
spp.), spiders, honey bees and blow flies being the most abundant taxa. These
results demonstrate that metabarcoding larval gut contents can be used to study the
Asian hornet diet and give a first snapshot of the prey items captured by V. v.
nigrithorax in the UK. This method could be used for future large-scale testing of
the gut contents of hornet nests, in order to provide a greater insight into the
foraging behaviour of this predator across Europe and elsewhere. Fera Science, The
National Agri-Food Innovation Campus, Sand Hutton, York YO41 1LZ, UK
 kirsty.stainton@nanoporetech.com; sam.mcgreig@fera.co.uk;
chris.conyers@fera.co.uk; sally.ponting@hotmail.co.uk; lee.butler@fera.co.uk;
paul.brown@fera.co.uk; eleanor.jones@fera.co.uk 10.3390/ani13030511 2023 13
3 - - 511 -
Delmiglio, Catia; Waite, David; Lilly, Sonia; Yan, Juncong; Elliott, Candace;
Pattemore, Julie; Guy, Paul; Thompson, Jeremy New Virus Diagnostic Approaches to
Ensuring the Ongoing Plant Biosecurity of Aotearoa New Zealand Viruses EN
Review Aotearoa; Māori; Oxford Nanopore; Illumina; Mickleham; post-entry
quarantine; point-of-use; environmental RNA/DNA To protect New Zealand’s
unique ecosystems and primary industries, imported plant materials must be
constantly monitored at the border for high-threat pathogens. Techniques adopted
for this purpose must be robust, accurate, rapid, and sufficiently agile to respond
to new and emerging threats. Polymerase chain reaction (PCR), especially real-time
PCR, remains an essential diagnostic tool but it is now being complemented by high-
throughput sequencing using both Oxford Nanopore and Illumina technologies,
allowing unbiased screening of whole populations. The demand for and value of
Point-of-Use (PoU) technologies, which allow for in situ screening, are also
increasing. Isothermal PoU molecular diagnostics based on recombinase polymerase
amplification (RPA) and loop-mediated amplification (LAMP) do not require expensive
equipment and can reach PCR-comparable levels of sensitivity. Recent advances in
PoU technologies offer opportunities for increased specificity, accuracy, and
sensitivities which makes them suitable for wider utilization by frontline or
border staff. National and international activities and initiatives are adopted to
improve both the plant virus biosecurity infrastructure and the integration,
development, and harmonization of new virus diagnostic technologies. Plant Health
and Environment Laboratory, Ministry for Primary Industries, P.O. Box 2095,
Auckland 1140, New Zealand catia.delmiglio@mpi.govt.nz; david.waite@mpi.govt.nz;
sonia.lilly@mpi.govt.nz; juncong.yan@mpi.govt.nz;
candace.elliott@agriculture.gov.au; julie.pattemore@agriculture.gov.au;
paul.guy@otago.ac.nz; jeremy.thompson@mpi.govt.nz 10.3390/v15020418 2023 15
2 - - 418 -
Vereecke, Nick; Woźniak, Aleksandra; Pauwels, Marthe; Coppens, Sieglinde; Nauwynck,
Hans; Cybulski, Piotr; Theuns, Sebastiaan; Stadejek, TomaszSuccessful Whole Genome
Nanopore Sequencing of Swine Influenza A Virus (swIAV) Directly from Oral Fluids
Collected in Polish Pig Herds Viruses EN Article epidemiology; nanopore
sequencing; sample storage; viral genomics; surveillance Influenza A virus (IAV)
is a single-stranded, negative-sense RNA virus and a common cause of seasonal flu
in humans. Its genome comprises eight RNA segments that facilitate reassortment,
resulting in a great variety of IAV strains. To study these processes, the genetic
code of each segment should be unraveled. Fortunately, new third-generation
sequencing approaches allow for cost-efficient sequencing of IAV segments.
Sequencing success depends on various factors, including proper sample storage and
processing. Hence, this work focused on the effect of storage of oral fluids and
swIAV sequencing. Oral fluids (n = 13) from 2017 were stored at −22 °C
and later transferred to −80 °C. Other samples (n = 21) were immediately
stored at −80 °C. A reverse transcription quantitative PCR (RT-qPCR) pre-
and post-storage was conducted to assess IAV viral loads. Next, samples were
subjected to two IAV long-read nanopore sequencing methods to evaluate success in
this complex matrix. A significant storage-associated loss of swIAV loads was
observed. Still, a total of 17 complete and 6 near-complete Polish swIAV genomes
were obtained. Genotype T, (H1avN2, seven herds), P (H1N1pdm09, two herds), U
(H1avN1, three herds), and A (H1avN1, 1 herd) were circulated on Polish farms. In
conclusion, oral fluids can be used for long-read swIAV sequencing when considering
appropriate storage and segment amplification protocols, which allows us to monitor
swIAV in an animal-friendly and cost-efficient manner. Laboratory of Virology,
Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium
nick.vereecke@ugent.be; aleksandra_wozniak@sggw.edu.pl;
marthe.pauwels@pathosense.com; sieglinde.coppens@pathosense.com;
hans.nauwynck@ugent.be; piotr.cybulski@goodvalley.com;
sebastiaan.theuns@pathosense.com; tomasz_stadejek@sggw.edu.pl 10.3390/v15020435
2023 15 2 - - 435 -
Rahman, Betul; Al-Marzooq, Farah; Saad, Hiba; Benzina, Dalenda; Al Kawas, Sausan
Dysbiosis of the Subgingival Microbiome and Relation to Periodontal Disease
in Association with Obesity and Overweight Nutrients EN Article
subgingival microbiome; oral dysbiosis; obesity; overweight; periodontitis;
16S rRNA sequencing Obesity causes gut dysbiosis; nevertheless, little is known
about the oral microbiome. We aimed to identify differences in the subgingival
microbiota influenced by body weight and periodontal status. Patients (n = 75)
recruited at the University Dental Hospital Sharjah, United Arab Emirates, were
distributed into three equal groups (healthy weight, overweight, and obese) sub-
divided into having either no-mild (NM) or moderate-severe (MS) periodontitis.
Subgingival plaques were collected. Microbiota were identified by 16S rRNA
sequencing using nanopore technology. Linear discriminant analysis demonstrated
significant bacterial biomarkers for body weight and periodontal health. Unique
microbiota signatures were identified, with enrichment of periopathogens in
patients with MS periodontitis (Aggregatibacter actinomycetemcomitans in obese,
Tannerella forsythia and Treponema denticola in overweight, Porphyromonas
gingivalis and Fusobacterium nucleatum in healthy weight), thus reflecting
differences in the microbiota affected by body weight. Other pathogenic bacteria,
such as Salmonella enterica and Klebsiella pneumoniae, were enriched in overweight
subjects with NM periodontitis, suggesting an increase in the relative abundance of
pathogens even in patients with good periodontal health if they were overweight.
Alpha and beta diversities were significantly different among the groups. Dysbiosis
of the subgingival microbiota in obese and overweight individuals was associated
with increased prevalence and severity of periodontal disease, which was correlated
with the body mass index. This study highlights the immense importance of the oral
microbiome and the need for lifestyle and dental interventions to resolve oral
dysbiosis and restore normal homeostasis. Department of Preventive and Restorative
Dentistry, College of Dental Medicine, University of Sharjah, Sharjah 27272, United
Arab Emirates brahman@sharjah.ac.ae; f.almarzooq@uaeu.ac.ae;
hisaad@sharjah.ac.ae; dalenda.benzina@gmail.com; sausan@sharjah.ac.ae
10.3390/nu15040826 2023 15 4 - - 826 -
Ooi, Mei; Trotter, Andrew; Smith, Gregory; Bridle, Andrew Characterisation of the
Gut Bacteria of Cultured and Wild Spiny Lobster Panulirus ornatus Applied
Microbiology EN Article gut bacteria; wild spiny lobster; cultured
spiny lobster; mussel; pellet; Vibrio The commercial onshore aquaculture of the
spiny lobster Panulirus ornatus, while in its infancy, has progressed rapidly from
the enabling research that continues at the University of Tasmania. The development
of lobster feeds, both fresh and manufactured, has been critical to the success of
this emerging aquaculture sector. Fresh feeds derived from mussel represent the
gold standard in terms of the growth performance of juvenile lobsters. Nonetheless,
concerns regarding availability, sustainability, and potential biosecurity issues
of fresh feeds highlight the importance of developing manufactured feeds for
lobster aquaculture. Wild lobsters are assumed to have a balanced natural diet that
allows for standard growth and development, and as such natural diets are often
used as a reference for feed development. Similarly, the gut microbiota associated
with a natural diet is assumed to reflect a healthy microbial assemblage. The aim
of this study was to compare the microbiota of the hindgut and hepatopancreas of
cultured P. ornatus fed with a commercial prawn pellet or mussel to that of wild
spiny lobster juveniles. Gut samples were analysed using Oxford Nanopore 16S rRNA
gene sequencing. Based on principal coordinate analysis, the gut bacteria of
cultured lobsters were different from the wild juveniles. The core microbiota of
the hindgut and hepatopancreas libraries were phyla Proteobacteria (Gamma, Alpha)
and Bacteroidetes. Vibrio was the most dominant genus in both organs. The
differences in bacterial relative abundance were mainly between cultured (pellet-,
mussel-fed) and wild lobsters. In conclusion, bacteria in the cultured lobsters had
significantly different profiles to that of the wild juveniles, indicating that
current onshore aquaculture practices alter the gut microbiota. A number of
different feeding and culture practices may be required if the aim of closed
culture practices is to attain a gut microbiota in cultured animals that is
representative of that found in wild spiny lobsters. Institute for Marine and
Antarctic Studies, University of Tasmania, Launceston, TAS 7250, Australia
mei.ooi@utas.edu.au; andrew.trotter@utas.edu.au; gregory.smith@utas.edu.au;
andrew.bridle@utas.edu.au 10.3390/applmicrobiol3010016 2023 3 1 -
- 16 -
Becker, Daniela; Popp, Denny; Bonk, Fabian; Kleinsteuber, Sabine; Harms, Hauke;
Centler, Florian Metagenomic Analysis of Anaerobic Microbial Communities Degrading
Short-Chain Fatty Acids as Sole Carbon Sources Microorganisms EN Article
anaerobic digestion; syntrophic acetate oxidation; syntrophic propionate
oxidation; syntrophic butyrate oxidation; methanogenic pathways; metagenome-
assembled genomes; hybrid assembly Analyzing microbial communities using
metagenomes is a powerful approach to understand compositional structures and
functional connections in anaerobic digestion (AD) microbiomes. Whereas short-read
sequencing approaches based on the Illumina platform result in highly fragmented
metagenomes, long-read sequencing leads to more contiguous assemblies. To evaluate
the performance of a hybrid approach of these two sequencing approaches we compared
the metagenome-assembled genomes (MAGs) resulting from five AD microbiome samples.
The samples were taken from reactors fed with short-chain fatty acids at different
feeding regimes (continuous and discontinuous) and organic loading rates (OLR).
Methanothrix showed a high relative abundance at all feeding regimes but was
strongly reduced in abundance at higher OLR, when Methanosarcina took over. The
bacterial community composition differed strongly between reactors of different
feeding regimes and OLRs. However, the functional potential was similar regardless
of feeding regime and OLR. The hybrid sequencing approach using Nanopore long-reads
and Illumina MiSeq reads improved assembly statistics, including an increase of the
N50 value (on average from 32 to 1740 kbp) and an increased length of the longest
contig (on average from 94 to 1898 kbp). The hybrid approach also led to a higher
share of high-quality MAGs and generated five potentially circular genomes while
none were generated using MiSeq-based contigs only. Finally, 27 hybrid MAGs were
reconstructed of which 18 represent potentially new species—15 of them
bacterial species. During pathway analysis, selected MAGs revealed similar gene
patterns of butyrate degradation and might represent new butyrate-degrading
bacteria. The demonstrated advantages of adding long reads to metagenomic analyses
make the hybrid approach the preferable option when dealing with complex
microbiomes. UFZ—Helmholtz Centre for Environmental Research, Department of
Environmental Microbiology, Permoserstr 15, 04318 Leipzig, Germany
daniela_becker91@gmx.de; denny.popp@medizin.uni-leipzig.de; wintersch-
fabian@web.de; sabine.kleinsteuber@ufz.de; hauke.harms@ufz.de; florian.centler@uni-
siegen.de 10.3390/microorganisms11020420 2023 11 2 - - 420
-
Kuzina, Ekaterina; Kislichkina, Angelina; Sizova, Angelika; Skryabin, Yury;
Novikova, Tatiana; Ershova, Olga; Savin, Ivan; Khokhlova, Olga; Bogun, Alexander;
Fursova, Nadezhda High-Molecular-Weight Plasmids Carrying Carbapenemase Genes
blaNDM-1, blaKPC-2, and blaOXA-48 Coexisting in Clinical Klebsiella pneumoniae
Strains of ST39 Microorganisms EN Article Klebsiella pneumoniae; hybrid
plasmid; carbapenemase; OXA-48; NDM-1; KPC-2; virulence genes Background:
Klebsiella pneumoniae, a member of the ESKAPE group of bacterial pathogens, has
developed multi-antimicrobial resistance (AMR), including resistance to
carbapenems, which has increased alarmingly due to the acquisition of carbapenemase
genes located on specific plasmids. Methods: Four clinical K. pneumoniae
isolates were collected from four patients of a neuro-intensive care unit in
Moscow, Russia, during the point prevalence survey. The AMR phenotype was estimated
using the Vitec-2 instrument, and whole genome sequencing (WGS) was done using
Illumina and Nanopore technologies. Results: All strains were resistant to beta-
lactams, nitrofurans, fluoroquinolones, sulfonamides, aminoglycosides, and
tetracyclines. WGS analysis revealed that all strains were closely related to
K. pneumoniae ST39, capsular type K-23, with 99.99% chromosome identity. The
novelty of the study is the description of the strains carrying simultaneously
three large plasmids of the IncHI1B, IncC, and IncFIB groups carrying the
carbapenemase genes of three types, blaOXA-48, blaNDM-1, and blaKPC-2,
respectively. The first of them, highly identical in all strains, was a hybrid
plasmid that combined two regions of the resistance genes (blaOXA-48 and blaTEM-1 +
blaCTX-M-15 + blaOXA-1 + catB + qnrS1 + int1) and a region of the virulence genes
(iucABCD, iutA, terC, and rmpA2::IS110). Conclusion: The spread of K. pneumoniae
strains carrying multiple plasmids conferring resistance even to last-resort
antibiotics is of great clinical concern. Department of Training and Improvement of
Specialists, State Research Center for Applied Microbiology and Biotechnology,
Territory “Kvartal A”, 142279 Obolensk, Russia e.leonova@mail.ru;
angelinakislichkina@yandex.ru; sizova1508@gmail.com; sjurikp@gmail.com;
pozitifka.15@yandex.ru; oershova@nsi.ru; savin@nsi.ru; khokhlovaol@mail.ru;
bogun@obolensk.org; n-fursova@yandex.ru 10.3390/microorganisms11020459 2023
11 2 - - 459 -
Lu, Zhuozhuang; Wang, Yongjin; Zou, Xiaohui; Hung, Tao Analysis of Fowl
Adenovirus 4 Transcriptome by De Novo ORF Prediction Based on Corrected Nanopore
Full-Length cDNA Sequencing Data Viruses EN Article fowl adenovirus 4;
transcriptome; full-length cDNA sequencing; RNA-seq; ORF prediction The
transcriptome of fowl adenovirus has not been comprehensively revealed. Here, we
attempted to analyze the fowl adenovirus 4 (FAdV-4) transcriptome by deep
sequencing. RNA samples were extracted from chicken LMH cells at 12, 18 or 26 h
post-FAdV-4 infection, and subjected to Illumina strand-specific RNA-seq or
nanopore full-length PCR-cDNA sequencing. After removing the reads of host cells,
the data of FAdV-4 nanopore full-length cDNAs (transcripts) were corrected with
reads from the Illumina RNA-seq, mapped to the viral genome and then used to
predict viral open reading frames (ORFs). Other than 42 known ORFs, 39 novel ORFs
were annotated to the FAdV-4 genome. Different from human adenovirus 5, one FAdV-4
ORF was often encoded by several transcripts, and more FAdV-4 ORFs were located on
two exons. With these data, 18 major transcription start sites and 15 major
transcription termination sites were defined, implying 18 viral promoters and 15
polyadenylation signals. The temporal cascade of viral gene transcription was
observed in FAdV-4-infected cells, with six promoters possessing considerable
activity in the early phase. Unexpectedly, four promoters, instead of one major
late promoter, were engaged in the transcription of the viral genus-common genes on
the forward strand. The clarification of the FAdV-4 transcriptome laid a solid
foundation for the study of viral gene function, virulence and virus evolution, and
it would help construct FAdV-4 as a gene transfer vehicle. The strategy of de novo
ORF prediction could be used to parse the transcriptome of other novel
adenoviruses. NHC Key Laboratory of Medical Virology and Viral Diseases,
National Institute for Viral Disease Control and Prevention, Chinese Center for
Disease Control and Prevention, Beijing 100052, China luzz@ivdc.chinacdc.cn;
wangyjwf@163.com; zouxh@ivdc.chinacdc.cn; hongt@cae.cn 10.3390/v15020529 2023
15 2 - - 529 -
MacKenzie, Morgan; Argyropoulos, Christos An Introduction to Nanopore Sequencing:
Past, Present, and Future Considerations Micromachines EN Review next-
generation sequencing; nanopore sequencing; biosensors; single-molecule analysis;
molecular diagnostics; genetics; transcriptomics; epigenetics There has been
significant progress made in the field of nanopore biosensor development and
sequencing applications, which address previous limitations that restricted
widespread nanopore use. These innovations, paired with the large-scale
commercialization of biological nanopore sequencing by Oxford Nanopore
Technologies, are making the platforms a mainstay in contemporary research
laboratories. Equipped with the ability to provide long- and short read sequencing
information, with quick turn-around times and simple sample preparation, nanopore
sequencers are rapidly improving our understanding of unsolved genetic,
transcriptomic, and epigenetic problems. However, there remain some key obstacles
that have yet to be improved. In this review, we provide a general introduction to
nanopore sequencing principles, discussing biological and solid-state nanopore
developments, obstacles to single-base detection, and library preparation
considerations. We present examples of important clinical applications to give
perspective on the potential future of nanopore sequencing in the field of
molecular diagnostics. Department of Internal Medicine, Division of Nephrology,
School of Medicine, University of New Mexico, Albuquerque, NM 87131, USA
memackenzie@unm.edu; cargyropoulos@salud.unm.edu 10.3390/mi14020459
2023 14 2 - - 459 -
Zhu, Liping; Gao, Xia; Zhang, Meihua; Hu, Chunhui; Yang, Wujie; Guo, Lizhong; Yang,
Song; Yu, Hailong; Yu, Hao Whole Genome Sequence of an Edible Mushroom
Oudemansiella raphanipes (Changgengu) Journal of Fungi EN Article
Oudemansiella raphanipes; genome; monokaryon; secondary metabolites; CAZyme;
phylogenetic analysis Oudemansiella raphanipes, considered as a well-known
culinary edible mushroom with a high content of natural bioactive substances, is
widely cultivated in China with the commercial name Changgengu. However, due to the
lack of genomic data, molecular and genetic study on O. raphanipes is rare. To
obtain a comprehensive overview of genetic characteristics and enhance the value of
O. raphanipes, two mating-compatible monokaryons isolated from the dikaryon were
applied for de novo genome sequencing and assembly using Nanopore and /or Illumina
sequencing platforms. One of the monokaryons, O. raphanipes CGG-A-s1, was annotated
with 21,308 protein-coding genes, of which 56 were predicted to be involved in the
biosynthesis of secondary metabolites such as terpene, type I PKS, NRPS, and
siderophore. Phylogenetic and comparative analysis of multiple fungi genomes
revealed a close evolutionary relationship between O. raphanipes and Mucidula mucid
based on single-copy orthologous protein genes. Significant collinearity was
detected between O. raphanipes and Flammulina velutipes on the synteny of inter-
species genomes. 664 CAZyme genes in CGG-A-s1 were identified with GHs and AAs
families significantly elevated when compared with the other 25 sequenced fungi,
indicating a strong wood degradation ability. Furthermore, the mating type locus
analysis revealed that CGG-A-s1 and CGG-A-s2 were conserved in the gene
organization of the mating A locus but various in that of the mating B locus. The
genome resource of O. raphanipes will provide new insights into its development of
genetic studies and commercial production of high-quality varieties. Shandong
Provincial Key Laboratory of Applied Mycology, School of Life Sciences, Qingdao
Agricultural University, 700 Changcheng Road, Chengyang District, Qingdao 266109,
China zhuliping1986@163.com; chuchugao@163.com; 20202106023@stu.qau.edu.cn;
201201012@qau.edu.cn; yangwujie.happy@163.com; glz119@126.com;
yangsong1209@163.com; yuhailong@saas.sh.cn; yuhao@qau.edu.cn 10.3390/jof9020266
2023 9 2 - - 266 -
Krasnikov, Nikita; Yuzhakov, Anton; Aliper, Taras; Gulyukin, Alexey Metagenomic
Approach Reveals the Second Subtype of PRRSV-1 in a Pathogen Spectrum during a
Clinical Outbreak with High Mortality in Western Siberia, Russia Viruses EN
Case Report porcine reproductive and respiratory syndrome; nanopore
sequencing; metagenomics; neurological disorders Porcine reproductive and
respiratory syndrome virus (PRRSV) has a significant economic impact on pig farming
worldwide by causing reproductive problems and affecting the respiratory systems of
swine. In Eastern Europe, PRRSV-1 strains are characterized by high genetic
variability, and pathogenicity differs among all known subtypes. This case study
describes the detection of a wide pathogen spectrum, including the second subtype
PRRSV-1, with a high mortality rate among nursery piglets (23.8%). This study was
conducted at a farrow-to-finish farm in the Western Siberia region of Russia.
Clinical symptoms included apathy, sneezing, and an elevation in body temperature,
and during the autopsy, degenerative lesions in different tissues were observed.
Moreover, 1.5 percent of the affected animals displayed clinical signs of the
central nervous system and were characterized by polyserositis. Nasal swabs from
diseased piglets and various tissue swabs from deceased animals were studied. For
diagnostics, the nanopore sequencing method was applied. All the samples tested
positive for PRRSV, and a more detailed analysis defined it as a second subtype of
PRRSV-1. The results, along with the clinical picture, showed a complex disease
etiology with the dominant role of PRRSV-1 and were informative about the high
pathogenicity of the subtype in question under field conditions. Federal State
Budget Scientific Institution “Federal Scientific Center VIEV”, 109428 Moscow,
Russia nick.krasnickoff2011@yandex.ru; anton_oskol@mail.ru; aliper@narvac.com;
admin@viev.ru 10.3390/v15020565 2023 15 2 - - 565 -
Ip, Hon; Uhm, Sarah; Killian, Mary; Torchetti, Mia An Evaluation of Avian
Influenza Virus Whole-Genome Sequencing Approaches Using Nanopore Technology
Microorganisms EN Article avian influenza; whole-genome sequencing;
next-generation sequencing; nanopore sequencing As exemplified by the global
response to the SARS-CoV-2 pandemic, whole-genome sequencing played an important
role in monitoring the evolution of novel viral variants and provided guidance on
potential antiviral treatments. The recent rapid and extensive introduction and
spread of highly pathogenic avian influenza virus in Europe, North America, and
elsewhere raises the need for similarly rapid sequencing to aid in appropriate
response and mitigation activities. To facilitate this objective, we investigate a
next-generation sequencing platform that uses a portable nanopore sequencing device
to generate and present data in real time. This platform offers the potential to
extend in-house sequencing capacities to laboratories that may otherwise lack
resources to adopt sequencing technologies requiring large benchtop instruments. We
evaluate this platform for routine use in a diagnostic laboratory. In this study,
we evaluate different primer sets for the whole genome amplification of influenza A
virus and evaluate five different library preparation approaches for sequencing on
the nanopore platform using the MinION flow cell. A limited amplification procedure
and a rapid procedure are found to be best among the approaches taken. National
Wildlife Health Center, U.S. Geological Survey, Department of the Interior,
Madison, WI 53711, USA hip@usgs.gov; uhm2@wisc.edu; mary.l.killian@usda.gov;
mia.kim.torchetti@usda.gov 10.3390/microorganisms11020529 2023 11 2
- - 529 -
Beikpour, Farzad; Pellegrini, Francesco; Lanave, Gianvito; Camero, Michele;
Catella, Cristiana; Di Martino, Barbara; Di Profio, Federica; Masotti, Chiara;
Battistini, Roberta; Serracca, Laura; La Rosa, Giuseppina; Martella, Vito;
Suffredini, Elisabetta Exploring the Astrovirome of Shellfish Matrices Using
Nanopore Sequencing Veterinary Sciences EN Article astrovirus;
shellfish; virome; enteric Astroviruses are important human enteric pathogens
transmissible with contaminated food and water. Astroviruses have also been
identified in mammals, birds, lower vertebrates and invertebrates. The genetic
diversity of human and animal astroviruses poses a challenge for diagnostics and
taxonomy. As a proof of concept, we used a panastrovirus consensus primer set, able
to amplify in a nested RT-PCR protocol a 400-nt-long fragment of the RNA-dependent
RNA polymerase of most members of the Astroviridae family, in conjunction with a
nanopore sequencing platform, to generate information on the astrovirome in filter-
feeding mollusks. Amplicons generated from bivalve samples were used to generate
libraries for deep sequencing. In three samples, only one unique RdRp sequence type
was obtained. However, in seven samples and in three barcodes with eleven pooled
samples, we identified a variety of known and unknown RdRp sequence types, in most
cases distantly related to astrovirus sequences available in the databases. In
total, 37 different sequence contigs were generated. Avian-origin astrovirus
sequences were predominant, likely due to contamination of shellfish harvesting
waters by marine birds. Astroviruses of the aquatic eco-system were also
identified, whereas human astroviruses were not detected. Department of Veterinary
Medicine, University of Bari Aldo Moro, 70010 Valenzano, Italy
farzad.beikpour@uniba.it; francesco.pellegrini@uniba.it;
gianvito.lanave@uniba.it; michele.camero@uniba.it; cristiana.catella@uniba.it;
bdimartino@unite.it; fdiprofio@unite.it; chiara.masotti@izsto.it;
roberta.battistini@izsto.it; laura.serracca@izsto.it; giuseppina.larosa@iss.it;
vito.martella@uniba.it; elisabetta.suffredini@iss.it 10.3390/vetsci10030175 2023
10 3 - - 175 -
Abou Kubaa, Raied; Amoia, Serafina; Altamura, Giuseppe; Minafra, Angelantonio;
Chiumenti, Michela; Cillo, Fabrizio Nanopore Technology Applied to Targeted
Detection of Tomato Brown Rugose Fruit Virus Allows Sequencing of Related Viruses
and the Diagnosis of Mixed Infections Plants EN Article high-
throughput sequencing (HTS); nanopore sequencing; tomato brown rugose fruit virus
(ToBRFV); tomato mottle mosaic virus (ToMMV); pepino mosaic virus (PepMV);
Tobamovirus; plant virus detection; diagnostics; mixed infections Tomato
(Solanum lycopersicum) plants from a commercial glasshouse were identified with
symptoms compatible with a tomato brown rugose fruit virus (ToBRFV) infection.
Reverse transcription-PCR and quantitative PCR confirmed the presence of ToBRFV.
Subsequently, the same RNA sample and a second from tomato plants infected with a
similar tobamovirus, tomato mottle mosaic virus (ToMMV), were extracted and
processed for high-throughput sequencing with the Oxford Nanopore Technology (ONT).
For the targeted detection of ToBRFV, the two libraries were synthesized by using
six ToBRFV sequence-specific primers in the reverse transcription step. This
innovative target enrichment technology enabled deep coverage sequencing of ToBRFV,
with 30% of the total reads mapping to the target virus genome and 57% mapping to
the host genome. The same set of primers applied to the ToMMV library generated 5%
of the total reads mapping to the latter virus, indicating that sequencing of
similar, non-target viral sequences was also allowed. Further, the complete genome
of pepino mosaic virus (PepMV) was also sequenced from the ToBRFV library, thus
suggesting that, even using multiple sequence-specific primers, a low rate of off-
target sequencing can usefully provide additional information on unexpected viral
species coinfecting the same samples in an individual assay. These results
demonstrate that targeted nanopore sequencing can specifically identify viral
agents and has sufficient sensitivity towards non-target organisms to provide
evidence of mixed virus infections. Institute for Sustainable Plant Protection—
National Research Council, 70126 Bari, Italy raied.aboukubaa@ipsp.cnr.it;
serena.amoia@ipsp.cnr.it; giuseppe.altamura@ipsp.cnr.it;
angelantonio.minafra@cnr.it; michela.chiumenti@ipsp.cnr.it; fabrizio.cillo@cnr.it
10.3390/plants12050999 2023 12 5 - - 999 -
López, Karla; Armijos, Carolina; Parra, Manuela; Torres, María The First Complete
Chloroplast Genome Sequence of Mortiño (Vaccinium floribundum) and
Comparative Analyses with Other Vaccinium Species Horticulturae EN
Article mortiño; Vaccinium floribundum; paramo; chloroplast genome;
comparative genomic analyses; Oxford Nanopore Technology (ONT) Vaccinium
floribundum, commonly known as mortiño, is a native high Andean wild species
of cultural and economic importance. Genomic resources for V. floribundum are
scarce, and a clear phylogenetic and evolutionary history for this species has yet
to be elucidated. This study aimed to assemble the complete chloroplast genome
sequence of this species and perform an in-depth comparative analysis with other
Vaccinium species. The chloroplast genome of V. floribundum was obtained using
Oxford Nanopore Technology (ONT). The de novo assembly of the chloroplast genome of
V. floribundum resulted in a 187,966 bp sequence, which contained 134 genes (84
Protein Coding Genes (PCGs), 42 transfer RNA (tRNA) genes, and 8 ribosomal RNA
(rRNA) genes). The comparative analysis of the V. floribundum chloroplast genome
with other nine chloroplast genomes of the Vaccinium species suggested that a
contraction/expansion event of the inverted repeat (IR) regions could have
occurred, causing the relocation of psbA and rpl32 genes. Additionally, a possible
loss of function of the ndhF gene was found. For the phylogenetic analysis based on
87 genes, the chloroplast genome of 19 species (including V. floribundum) was used
and revealed that V. myrtillus could be a sister group of V. floribundum.
Altogether, our findings provide insights into the plastome characteristics and the
phylogeny of V. floribundum. This study describes the complete chloroplast genome
sequence of V. floribundum as the first genomic resource available for an Andean
species native to Ecuador. Laboratorio de Biotecnología de Plantas, Universidad
San Francisco de Quito (USFQ), Diego de Robles y Vía Interoceánica, Quito 170901,
Ecuador krojas@usfq.edu.ec; carmijos@usfq.edu.ec; mparrav@usfq.edu.ec;
ltorres@usfq.edu.ec 10.3390/horticulturae9030302 2023 9 3 - -
302 -
Zou, Yuchen; Guo, Qing; Chang, Yidan; Zhong, Yongyong; Cheng, Lin; Wei, Wei
Effects of Maternal High-Fructose Diet on Long Non-Coding RNAs and Anxiety-
like Behaviors in Offspring International Journal of Molecular Sciences EN
Article gestation; lactation; brain development; full-length RNA
sequencing; Oxford Nanopore Technologies Increased fructose intake is an
international issue. A maternal high-fructose diet during gestation and lactation
could affect nervous system development in offspring. Long non-coding RNA (lncRNA)
plays an important role in brain biology. However, the mechanism whereby maternal
high-fructose diets influence offspring brain development by affecting lncRNAs is
still unclear. Here, we administered 13% and 40% fructose water to establish a
maternal high-fructose diet model during gestation and lactation. To determine
lncRNAs and their target genes, full-length RNA sequencing was performed using the
Oxford Nanopore Technologies platform, and 882 lncRNAs were identified. Moreover,
the 13% fructose group and the 40% fructose group had differentially expressed
lncRNA genes compared with the control group. Enrichment analyses and co-expression
analyses were performed to investigate the changes in biological function.
Furthermore, enrichment analyses, behavioral science experiments, and molecular
biology experiments all indicated that the fructose group offspring showed anxiety-
like behaviors. In summary, this study provides insight into the molecular
mechanisms underlying maternal high-fructose diet-induced lncRNA expression and co-
expression of lncRNA and mRNA. Child and Adolescent Health, School of Public
Health, China Medical University, No. 77 Puhe Road, Shenyang North New Area,
Shenyang 110122, China yczou@cmu.edu.cn; qingguo@cmu.edu.cn;
changyidan6090@163.com; zhongyoyo925@163.com; chenglin99990@163.com;
wwei@cmu.edu.cn 10.3390/ijms24054460 2023 24 5 - - 4460 -
Nazarenko, Maria; Sleptcov, Aleksei; Zarubin, Aleksei; Salakhov, Ramil; Shevchenko,
Alexander; Tmoyan, Narek; Elisaphenko, Eugeny; Zubkova, Ekaterina; Zheltysheva,
Nina; Ezhov, Marat; Kukharchuk, Valery; Parfyonova, Yelena; Zakian, Suren;
Zakharova, Irina Calling and Phasing of Single-Nucleotide and Structural Variants
of the LDLR Gene Using Oxford Nanopore MinION International Journal of Molecular
Sciences EN Article LDLR; Oxford Nanopore; familial hypercholesterolemia;
structural variant; haplotype The LDLR locus has clinical significance for lipid
metabolism, Mendelian familial hypercholesterolemia (FH), and common lipid
metabolism-related diseases (coronary artery disease and Alzheimer’s
disease), but its intronic and structural variants are underinvestigated. The aim
of this study was to design and validate a method for nearly complete sequencing of
the LDLR gene using long-read Oxford Nanopore sequencing technology (ONT). Five PCR
amplicons from LDLR of three patients with compound heterozygous FH were analyzed.
We used standard workflows of EPI2ME Labs for variant calling. All rare missense
and small deletion variants detected previously by massively parallel sequencing
and Sanger sequencing were identified using ONT. One patient had a 6976 bp deletion
(exons 15 and 16) that was detected by ONT with precisely located breakpoints
between AluY and AluSx1. Trans-heterozygous associations between mutation
c.530C>T and c.1054T>C, c.2141-966_2390-330del, and c.1327T>C, and between
mutations c.1246C>T and c.940+3_940+6del of LDLR, were confirmed. We
demonstrated the ability of ONT to phase variants, thereby enabling haplotype
assignment for LDLR with personalized resolution. The ONT-based method was able to
detect exonic variants with the additional benefit of intronic analysis in one run.
This method can serve as an efficient and cost-effective tool for diagnosing FH and
conducting research on extended LDLR haplotype reconstruction. Research Institute
of Medical Genetics, Tomsk National Research Medical Center, Russian Academy of
Sciences, Tomsk 634050, Russia maria.nazarenko@medgenetics.ru;
alexei.sleptcov@medgenetics.ru; a.a.zarubin@gmail.com;
ramil.salakhov@medgenetics.ru; epigene@bionet.nsc.ru; ntmoyan@gmail.com;
antares@bionet.nsc.ru; cat.zubkova@gmail.com; n.zheltysheva@g.nsu.ru;
marat_ezhov@mail.ru; v_kukharch@mail.ru; yeparfyon@mail.ru; zakian@bionet.nsc.ru;
zakharova@bionet.nsc.ru 10.3390/ijms24054471 2023 24 5 - - 4471
-
Zhang, Liguo; Bisht, Punam; Flamier, Anthony; Barrasa, M.; Friesen, Max; Richards,
Alexsia; Hughes, Stephen; Jaenisch, Rudolf LINE1-Mediated Reverse
Transcription and Genomic Integration of SARS-CoV-2 mRNA Detected in Virus-Infected
but Not in Viral mRNA-Transfected Cells Viruses EN Article SARS-CoV-2;
LINE1; retrotransposition; WGS; enrichment sequencing; RNA transfection SARS-
CoV-2 sequences can be reverse-transcribed and integrated into the genomes of
virus-infected cells by a LINE1-mediated retrotransposition mechanism. Whole-genome
sequencing (WGS) methods detected retrotransposed SARS-CoV-2 subgenomic sequences
in virus-infected cells overexpressing LINE1, while an enrichment method (TagMap)
identified retrotranspositions in cells that did not overexpress LINE1. LINE1
overexpression increased retrotranspositions about 1000-fold as compared to non-
overexpressing cells. Nanopore WGS can directly recover retrotransposed viral and
flanking host sequences, but its sensitivity depends on the depth of sequencing (a
typical 20-fold sequencing depth would only examine 10 diploid cell equivalents).
In contrast, TagMap enriches the host–virus junctions and can interrogate up
to 20,000 cells and is able to detect rare viral retrotranspositions in LINE1 non-
overexpressing cells. Although Nanopore WGS is 10–20-fold more sensitive per
tested cell, TagMap can interrogate 1000–2000-fold more cells and, therefore,
can identify infrequent retrotranspositions. When comparing SARS-CoV-2 infection
and viral nucleocapsid mRNA transfection by TagMap, retrotransposed SARS-CoV-2
sequences were only detected in infected but not in transfected cells.
Retrotransposition in virus-infected cells, in contrast to transfected cells, may
be facilitated because virus infection, in contrast to viral RNA transfection,
results in significantly higher viral RNA levels and stimulates LINE1 expression by
causing cellular stress. Whitehead Institute for Biomedical Research,
Cambridge, MA 02142, USA liguo@wi.mit.edu; punamb@wi.mit.edu;
aflamier@wi.mit.edu; ibarrasa@wi.mit.edu; mfriesen@wi.mit.edu; arichard@wi.mit.edu;
hughesst@mail.nih.gov; jaenisch@wi.mit.edu 10.3390/v15030629 2023 15 3
- - 629 -
Sigova, Elizaveta; Pushkova, Elena; Rozhmina, Tatiana; Kudryavtseva, Ludmila;
Zhuchenko, Alexander; Novakovskiy, Roman; Zhernova, Daiana; Povkhova, Liubov;
Turba, Anastasia; Borkhert, Elena; Melnikova, Nataliya; Dmitriev, Alexey;
Dvorianinova, Ekaterina Assembling Quality Genomes of Flax Fungal Pathogens from
Oxford Nanopore Technologies Data Journal of Fungi EN Article
Aureobasidium pullulans; Colletotrichum lini; Fusarium verticillioides;
Fusarium moniliforme; pathogens; flax; nanopore sequencing; genome assembly Flax
(Linum usitatissimum L.) is attacked by numerous devastating fungal pathogens,
including Colletotrichum lini, Aureobasidium pullulans, and Fusarium
verticillioides (Fusarium moniliforme). The effective control of flax diseases
follows the paradigm of extensive molecular research on pathogenicity. However,
such studies require quality genome sequences of the studied organisms. This
article reports on the approaches to assembling a high-quality fungal genome from
the Oxford Nanopore Technologies data. We sequenced the genomes of C. lini, A.
pullulans, and F. verticillioides (F. moniliforme) and received different volumes
of sequencing data: 1.7 Gb, 3.9 Gb, and 11.1 Gb, respectively. To obtain the
optimal genome sequences, we studied the effect of input data quality and genome
coverage on assembly statistics and tested the performance of different assembling
and polishing software. For C. lini, the most contiguous and complete assembly was
obtained by the Flye assembler and the Homopolish polisher. The genome coverage had
more effect than data quality on assembly statistics, likely due to the relatively
low amount of sequencing data obtained for C. lini. The final assembly was 53.4 Mb
long and 96.4% complete (according to the glomerellales_odb10 BUSCO dataset),
consisted of 42 contigs, and had an N50 of 4.4 Mb. For A. pullulans and F.
verticillioides (F. moniliforme), the best assemblies were produced by
Canu–Medaka and Canu–Homopolish, respectively. The final assembly of A.
pullulans had a length of 29.5 Mb, 99.4% completeness (dothideomycetes_odb10), an
N50 of 2.4 Mb and consisted of 32 contigs. F. verticillioides (F. moniliforme)
assembly was 44.1 Mb long, 97.8% complete (hypocreales_odb10), consisted of 54
contigs, and had an N50 of 4.4 Mb. The obtained results can serve as a guideline
for assembling a de novo genome of a fungus. In addition, our data can be used in
genomic studies of fungal pathogens or plant–pathogen interactions and assist
in the management of flax diseases. Engelhardt Institute of Molecular Biology,
Russian Academy of Sciences, Moscow 119991, Russia sigova.ea@phystech.edu;
pushkova18@gmail.com; tatyana_rozhmina@mail.ru; lpkudryavtseva@icloud.com;
ecovilar@mail.ru; 0legovich46@mail.ru; zhernova.d@yandex.ru;
povhova.lv@phystech.edu; anastas.turba@gmail.com; sashai@inbox.ru; mnv-
4529264@yandex.ru; alex_245@mail.ru; dvorianinova.em@phystech.edu
10.3390/jof9030301 2023 9 3 - - 301 -
Kruse, Elisabeth; Göringer, H. Nanopore-Based Direct RNA Sequencing of the
Trypanosoma brucei Transcriptome Identifies Novel lncRNAs Genes EN Article
direct RNA sequencing; long-read RNA sequencing; nanopore sequencing;
transcriptome analysis; African trypanosomes; long noncoding RNAs; bloodstream-
stage trypanosomes; insect-stage trypanosomes Trypanosomatids are single-cell
eukaryotic parasites. Unlike higher eukaryotes, they control gene expression post-
transcriptionally and not at the level of transcription initiation. This involves
all known cellular RNA circuits, from mRNA processing to mRNA decay, to
translation, in addition to a large panel of RNA-interacting proteins that modulate
mRNA abundance. However, other forms of gene regulation, for example by lncRNAs,
cannot be excluded. LncRNAs are poorly studied in trypanosomatids, with only a
single lncRNA characterized to date. Furthermore, it is not clear whether the
complete inventory of trypanosomatid lncRNAs is known, because of the inherent
cDNA-recoding and DNA-amplification limitations of short-read RNA sequencing. Here,
we overcome these limitations by using long-read direct RNA sequencing (DRS) on
nanopore arrays. We analyze the native RNA pool of the two main lifecycle stages of
the African trypanosome Trypanosoma brucei, with a special emphasis on the
inventory of lncRNAs. We identify 207 previously unknown lncRNAs, 32 of which are
stage-specifically expressed. We also present insights into the complexity of the
T. brucei transcriptome, including alternative transcriptional start and stop sites
and potential transcript isoforms, to provide a bias-free understanding of the
intricate RNA landscape in T. brucei. Molecular Genetics, Technical University
Darmstadt, Schnittspahnstr. 10, 64287 Darmstadt, Germany kruse@bio.tu-
darmstadt.de; goringer@bio.tu-darmstadt.de 10.3390/genes14030610 2023 14
3 - - 610 -
Cifuentes, Rosa; Padilla, José; de la Morena-Barrio, María; de la Morena-Barrio,
Belén; Bravo-Pérez, Carlos; Garrido-Rodríguez, Pedro; Llamas, María; Miñano,
Antonia; Vicente, Vicente; Lozano, María; Corral, Javier Usefulness and
Limitations of Multiple Ligation-Dependent Probe Amplification in Antithrombin
Deficiency International Journal of Molecular Sciences EN Article
antithrombin deficiency; multiplex ligation-dependent probe amplification;
structural variants; genetic variants Multiplex ligation-dependent probe
amplification (MLPA) identifies genetic structural variants in SERPINC1 in 5% of
cases with antithrombin deficiency (ATD), the most severe congenital thrombophilia.
Our aim was to unravel the utility and limitations of MLPA in a large cohort of
unrelated patients with ATD (N = 341). MLPA identified 22 structural variants (SVs)
causing ATD (6.5%). MLPA did not detect SVs affecting introns (four cases), and the
diagnosis was inaccurate in two cases according to long-range PCR or nanopore
sequencing. MLPA was used to detect possible hidden SVs in 61 cases with type I
deficiency with single nucleotide variations (SNVs) or small insertion/deletion
(INDEL). One case had a false deletion of exon 7, as the 29-bp deletion affected an
MLPA probe. We evaluated 32 variants affecting MLPA probes: 27 SNVs and 5 small
INDELs. In three cases, MLPA gave false-positive results, all diagnosed as
deletions of the affected exon: a small INDEL complex, and two SNVs affecting MLPA
probes. Our study confirms the utility of MLPA to detect SVs in ATD, but also shows
some limitations in detecting intronic SVs. MLPA renders imprecise and false-
positive results for genetic defects which affect MLPA probes. Our results
encourage the validation of MLPA results. Servicio de Hematología y Oncología
Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación,
Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER, 30008 Murcia, Spain
rcifuentesriquelme@gmail.com; josepadillaruizcrh@gmail.com;
uge2985@hotmail.com; belendelamorenabarrio@gmail.com; carlos.bravop@um.es;
pedro.garridor@outlook.es; llamasmaria999@gmail.com; antoniadpminano@gmail.com;
vicente.vicente@carm.es; mllozano@um.es; javiercorraldelacalle@gmail.com
10.3390/ijms24055023 2023 24 5 - - 5023 -
Cruz-Silva, Ana; Laureano, Gonçalo; Pereira, Marcelo; Dias, Ricardo; Silva, José;
Oliveira, Nuno; Gouveia, Catarina; Cruz, Cristina; Gama-Carvalho, Margarida;
Alagna, Fiammetta; Duarte, Bernardo; Figueiredo, Andreia A New Perspective for
Vineyard Terroir Identity: Looking for Microbial Indicator Species by Long Read
Nanopore Sequencing Microorganisms EN Article soil metagenomic;
microbiome; long-read nanopore sequencing; microbial signature; grapevine
Grapevine is one of the most important fruit crops worldwide, being Portugal
one of the top wine producers. It is well established that wine sensory
characteristics from a particular region are defined by the physiological responses
of the grapevine to its environment and thus, the concept of terroir in viticulture
was established. Among all the factors that contribute to terroir definition, soil
microorganisms play a major role from nutrient recycling to a drastic influence on
plant fitness (growth and protection) and of course wine production. Soil
microbiome from four different terroirs in Quinta dos Murças vineyard was
analysed through long-read Oxford Nanopore sequencing. We have developed an
analytical pipeline that allows the identification of function, ecologies, and
indicator species based on long read sequencing data. The Douro vineyard was used
as a case study, and we were able to establish microbiome signatures of each
terroir. Biosystems & Integrative Sciences Institute (BioISI), Faculdade de
Ciências da Universidade de Lisboa, 1749-016 Lisboa, Portugal amcsilva@fc.ul.pt;
gmlaureano@fc.ul.pt; mlpereira@fc.ul.pt; rpdias@fc.ul.pt; jose.luis@esporao.com;
nuno.oliveira@nbi.pt; cagouveia@fc.ul.pt; ccruz@fc.ul.pt; mhcarvalho@fc.ul.pt;
fiammetta.alagna@enea.it; baduarte@fc.ul.pt; aafigueiredo@fc.ul.pt
10.3390/microorganisms11030672 2023 11 3 - - 672 -
Iurescia, Manuela; Diaconu, Elena; Alba, Patricia; Feltrin, Fabiola; Buccella,
Carmela; Onorati, Roberta; Giacomi, Angelo; Caprioli, Andrea; Franco, Alessia;
Battisti, Antonio; Carfora, Virginia Genomics Insight into cfr-Mediated
Linezolid-Resistant LA-MRSA in Italian Pig Holdings Antibiotics EN Article
Staphylococcus aureus; LA-MRSA; linezolid; cfr; WGS; long reads; short reads;
bioinformatics analysis; CC1; CC398 The cfr genes encode for a 23S rRNA
methyltransferase, conferring a multiresistance phenotype to phenicol, lincosamide,
oxazolidinone, pleuromutilin, and streptogramin A antibiotics. These genes have
been described in staphylococci, including methicillin-resistant Staphylococcus
aureus (MRSA). In this study, we retrospectively performed an in-depth genomic
characterisation of three cfr-positive, multidrug-resistant (MDR) livestock-
associated (LA) MRSA clonal complexes (CCs) 1 and 398 detected in different Italian
pig holdings (2008–2011) during population studies on Italian livestock
(2008–2014). We used a combined Illumina and Oxford Nanopore Technologies
(ONT) whole genome sequencing (WGS) approach on two isolates (the 2008 CC1 and the
2010 CC398 isolates, but not the 2011 CC1 isolate). Interestingly, the three
isolates presented different cfr variants, with only one displaying a linezolid-
resistant phenotype. In isolate 2008 CC1, the cfr gene was identified within a
Tn558 composite transposon-like structure flanked by IS elements located on a novel
44,826 bp plasmid. This represents the first report of CC1 LA-MRSA harbouring the
cfr gene in its functional variant. Differently, cfr was chromosomally located in
isolate 2010 CC398. Our findings have significant public health implications,
confirm the need for the continuous genomic surveillance of cfr-positive zoonotic
LA-MRSA, and backdate cfr presence in LA-MRSA from Italian pigs to at least 2008.
National Reference Laboratory for Antimicrobial Resistance, Department of
General Diagnostics, Istituto Zooprofilattico Sperimentale del Lazio e Della
Toscana “M. Aleandri”, 00178 Rome, Italy manuela.iurescia@izslt.it;
elena.diaconu@izslt.it; patricia.alba@izslt.it; fabiola.feltrin@izslt.it;
carmela.buccella@izslt.it; roberta.onorati@izslt.it; angelo.giacomi@izslt.it;
andrea.caprioli@izslt.it; alessia.franco@izslt.it; antonio.battisti@izslt.it;
virginia.carfora@izslt.it 10.3390/antibiotics12030530 2023 12 3 -
- 530 -
Klink, Amy; Rula, Oleksandr; Sushko, Mykola; Bezymennyi, Maksym; Mezinov,
Oleksandr; Gaidash, Oleksandr; Bai, Xiao; Stegniy, Anton; Sapachova, Maryna;
Datsenko, Roman; Skorokhod, Sergiy; Nedosekov, Vitalii; Hill, Nichola; Ninua,
Levan; Kovalenko, Ganna; Ducluzeau, Anne; Mezhenskyi, Andriy; Buttler, Jeremy;
Drown, Devin; Causey, Douglas; Stegniy, Borys; Gerilovych, Anton; Bortz, Eric;
Muzyka, Denys Discovery of Avian Paramyxoviruses APMV-1 and APMV-6 in
Shorebirds and Waterfowl in Southern Ukraine Viruses EN Article viral
ecology; surveillance of avian paramyxoviruses; APMV; wild birds; next-generation
sequencing; minion; Azov-Black Sea region in Ukraine Emerging RNA virus infections
are a growing concern among domestic poultry industries due to the severe impact
they can have on flock health and economic livelihoods. Avian paramyxoviruses
(APMV; avulaviruses, AaV) are pathogenic, negative-sense RNA viruses that cause
serious infections in the respiratory and central nervous systems. APMV was
detected in multiple avian species during the 2017 wild bird migration season in
Ukraine and studied using PCR, virus isolation, and sequencing. Of 4090 wild bird
samples collected, mostly from southern Ukraine, eleven isolates were grown in ovo
and identified for APMV serotype by hemagglutinin inhibition test as: APMV-1, APMV-
4, APMV-6, and APMV-7. To build One Health’s capacity to characterize APMV
virulence and analyze the potential risks of spillover to immunologically
naïve populations, we sequenced virus genomes in veterinary research labs in
Ukraine using a nanopore (MinION) platform. RNA was extracted and amplified using a
multiplex tiling primer approach to specifically capture full-length APMV-1 (n = 5)
and APMV-6 (n = 2) genomes at high read depth. All APMV-1 and APMV-6 fusion (F)
proteins possessed a monobasic cleavage site, suggesting these APMVs were likely
low virulence, annually circulating strains. Utilization of this low-cost method
will identify gaps in viral evolution and circulation in this understudied but
important critical region for Eurasia. Department of Biological Sciences,
University of Alaska Anchorage, Anchorage, AK 99508, USA amy.klink@unlv.edu;
aleksrula75@gmail.com; m.i.sushko@gmail.com; nomax@ukr.net; mezinov.alex@gmail.com;
alexgaidash@gmail.com; xbai715@gmail.com; antonborisovich@gmail.com;
m_sapacheva@meta.ua; rozariogro@bigmir.net; skorohodsv@gmail.com;
nedosekov06@gmail.com; nichola.hill@umb.edu; levan.ninua@iliauni.edu.ge;
ak2388@cam.ac.uk; alducluzeau@gmail.com; mezhaavet@gmail.com; jdbuttler@alaska.edu;
dmdrown@alaska.edu; dcausey@alaska.edu; boris.stegniy@gmail.com;
antger2011@gmail.com; ebortz@alaska.edu; dmuzyka77@gmail.com 10.3390/v15030699
2023 15 3 - - 699 -
Yang, Heyu; Chen, Haimei; Ni, Yang; Li, Jingling; Cai, Yisha; Wang, Jiehua; Liu,
Chang Mitochondrial Genome Sequence of Salvia officinalis (Lamiales: Lamiaceae)
Suggests Diverse Genome Structures in Cogeneric Species and Finds the Stop Gain of
Genes through RNA Editing Events International Journal of Molecular Sciences
EN Article Salvia officinalis; Lamiales; mitogenome; multi-chromosomal
structure; introns Our previous study was the first to confirm that the
predominant conformation of mitochondrial genome (mitogenome) sequence of Salvia
species contains two circular chromosomes. To further understand the organization,
variation, and evolution of Salvia mitogenomes, we characterized the mitogenome of
Salvia officinalis. The mitogenome of S. officinalis was sequenced using Illumina
short reads and Nanopore long reads and assembled using a hybrid assembly strategy.
We found that the predominant conformation of the S. officinalis mitogenome also
had two circular chromosomes that were 268,341 bp (MC1) and 39,827 bp (MC2) in
length. The S. officinalis mitogenome encoded an angiosperm-typical set of 24 core
genes, 9 variable genes, 3 rRNA genes, and 16 tRNA genes. We found many
rearrangements of the Salvia mitogenome through inter- and intra-specific
comparisons. A phylogenetic analysis of the coding sequences (CDs) of 26 common
protein-coding genes (PCGs) of 11 Lamiales species and 2 outgroup taxa strongly
indicated that the S. officinalis was a sister taxon to S. miltiorrhiza, consistent
with the results obtained using concatenated CDs of common plastid genes. The
mapping of RNA-seq data to the CDs of PCGs led to the identification of 451 C-to-U
RNA editing sites from 31 PCGs of the S. officinalis mitogenome. Using PCR
amplification and Sanger sequencing methods, we successfully validated 113 of the
126 RNA editing sites from 11 PCGs. The results of this study suggest that the
predominant conformation of the S. officinalis mitogenome are two circular
chromosomes, and the stop gain of rpl5 was found through RNA editing events of the
Salvia mitogenome. School of Environmental Science and Engineering, Tianjin
University, Tianjin 300072, China heyuyang@tju.edu.cn; hmchen@implad.ac.cn;
ny_work@126.com; lijingling1997@163.com; caiyisha198999@163.com;
jiehuawang@tju.edu.cn; cliu@implad.ac.cn 10.3390/ijms24065372 2023 24 6
- - 5372 -
Pascucci, Giuseppe; Morrocchi, Elena; Pighi, Chiara; Rotili, Arianna; Neri,
Alessia; Medri, Chiara; Olivieri, Giulio; Sanna, Marco; Rasi, Gianmarco; Persaud,
Deborah; Chahroudi, Ann; Lichterfeld, Mathias; Nastouli, Eleni; Cancrini, Caterina;
Amodio, Donato; Rossi, Paolo; Cotugno, Nicola; Palma, PaoloHow CD4+ T Cells
Transcriptional Profile Is Affected by Culture Conditions: Towards the Design of
Optimal In Vitro HIV Reactivation Assays Biomedicines EN Article T cell
activation; in vitro cultures; HIV reactivation; RNA sequencing; Oxford Nanopore
technologies; autologous plasma; TCR signaling cascade; PMA/ionomycin stimulation;
CD4+ T cells; RPMI Most of the current assays directed at the investigation of
HIV reactivation are based on cultures of infected cells such as Peripheral Blood
Mononuclear Cells (PBMCs) or isolated CD4+ T cells, stimulated in vitro with
different activator molecules. The culture media in these in vitro tests lack many
age- and donor-specific immunomodulatory components normally found within the
autologous plasma. This triggered our interest in understanding the impact that
different matrices and cell types have on T cell transcriptional profiles following
in vitro culture and stimulation. Methods: Unstimulated or stimulated CD4+ T cells
of three young adults with perinatal HIV-infection were isolated from PBMCs before
or after culture in RPMI medium or autologous plasma. Transcriptomes were sequenced
using Oxford Nanopore technologies. Results: Transcriptional profiles revealed the
activation of similar pathways upon stimulation in both media with a higher
magnitude of TCR cascade activation in CD4+ lymphocytes cultured in RPMI.
Conclusions: These results suggest that for studies aiming at quantifying the
magnitude of biological mechanisms under T cell activation, the autologous plasma
could better approximate the in vivo environment. Conversely, if the study aims at
defining qualitative aspects, then RPMI culture could provide more evident results.
Research Unit of Clinical Immunology and Vaccinology, Bambino Gesù Children’s
Hospital, 00165 Rome, Italy grubens.pascucci@opbg.net; elena.morrocchi@opbg.net;
chiara.pighi@opbg.net; arianna.rotili@hotmail.it; alessia.neri@opbg.net;
chiara.medri@opbg.net; giulio.olivieri@opbg.net; marco.sanna@opbg.net;
gianmarco.rasi@gmail.com; dpers@jhmi.edu; ann.m.chahroudi@emory.edu;
mlichterfeld@mgh.harvard.edu; e.nastouli@ucl.ac.uk; cancrini@med.uniroma2.it;
donato.amodio@opbg.net; paolo.rossi@opbg.net; nicola.cotugno@opbg.net;
paolo.palma@opbg.net 10.3390/biomedicines11030888 2023 11 3 - -
888 -
Cordeiro, Daniela; Camelo, Alexandra; Pedrosa, Ana; Brandão, Inês; Canhoto, Jorge;
Espírito Santo, Christophe; Correia, Sandra An Efficient Method to Prepare
Barcoded cDNA Libraries from Plant Callus for Long-Read Sequencing Methods and
Protocols EN Protocol amplification-free protocol; direct cDNA sequencing;
high-throughput sequencing; MinION; Oxford Nanopore Technologies®; poly(A) RNA;
Solanaceae; Solanum betaceum; transcriptome; woody plant Long-read sequencing
methods allow a comprehensive analysis of transcriptomes in identifying full-length
transcripts. This revolutionary method represents a considerable breakthrough for
non-model species since it allows enhanced gene annotation and gene expression
studies when compared to former sequencing methods. However, woody plant tissues
are challenging to the successful preparation of cDNA libraries, thus, impairing
further cutting-edge sequencing analyses. Here, a detailed protocol for preparing
cDNA libraries suitable for high throughput RNA sequencing using Oxford Nanopore
Technologies® is described. This method was used to prepare eight barcoded cDNA
libraries from two Solanum betaceum cell lines: one with compact morphology and
embryogenic competency (EC) and another with friable and non-embryogenic (NEC). The
libraries were successfully sequenced, and data quality assessment showed high mean
quality scores. Using this method, long-read sequencing will allow a comprehensive
analysis of plant transcriptomes. Centre for Functional Ecology, TERRA Associate
Laboratory, Department of Life Sciences, University of Coimbra, Calçada Martim de
Freitas, 3000-456 Coimbra, Portugal danielacordeiro@outlook.pt;
alexandra.camelo@cataa.pt; anasimoespedrosa@gmail.com; inesm.brandao@gmail.com;
jorgecan@uc.pt; cespiritosanto@cataa.pt; sandraimc@uc.pt 10.3390/mps6020031
2023 6 2 - - 31 -
Szoboszlay, Márton; Schramm, Laetitia; Pinzauti, David; Scerri, Jeanesse;
Sandionigi, Anna; Biazzo, Manuele Nanopore Is Preferable over Illumina for 16S
Amplicon Sequencing of the Gut Microbiota When Species-Level Taxonomic
Classification, Accurate Estimation of Richness, or Focus on Rare Taxa Is Required
Microorganisms EN Article Nanopore; Illumina; 16S rRNA; gut
microbiota; species-level taxonomy Nanopore sequencing is a promising technology
used for 16S rRNA gene amplicon sequencing as it can provide full-length 16S reads
and has a low up-front cost that allows research groups to set up their own
sequencing workflows. To assess whether Nanopore with the improved error rate of
the Kit 12 chemistry should be adopted as the preferred sequencing technology
instead of Illumina for 16S amplicon sequencing of the gut microbiota, we used a
mock community and human faecal samples to compare diversity, richness, and
species-level community structure, as well as the replicability of the results.
Nanopore had less noise, better accuracy with the mock community, a higher
proportion of reads from the faecal samples classified to species, and better
replicability. The difference between the Nanopore and Illumina results of the
faecal bacterial community structure was significant but small compared to the
variation between samples. The results show that Nanopore is a better choice for
16S rRNA gene amplicon sequencing when the focus is on species-level taxonomic
resolution, the investigation of rare taxa, or an accurate estimation of richness.
Illumina 16S sequencing should be reserved for communities with many unknown
species, and for studies that require the resolution of amplicon sequence variants.
The BioArte Ltd., SGN 3000 San Gwann, Malta m.szoboszlay@thebioarte.com;
l.schramm@thebioarte.com; d.pinzauti@thebioarte.com; j.scerri@thebioarte.com;
anna.sandionigi@unimib.it; m.biazzo@thebioarte.com
10.3390/microorganisms11030804 2023 11 3 - - 804 -
Al-Trad, Esra’a; Che Hamzah, Ainal; Puah, Suat; Chua, Kek; Hanifah, Muhamad; Ayub,
Qasim; Palittapongarnpim, Prasit; Kwong, Stephen; Chew, Ching; Yeo, Chew
Complete Genome Sequence and Analysis of a ST573 Multidrug-Resistant
Methicillin-Resistant Staphylococcus aureus SauR3 Clinical Isolate from Terengganu,
Malaysia Pathogens EN Article Staphylococcus aureus ST573; hybrid
assembly; resistance genes; SCCmec element; plasmids; genomic islands; prophages
Methicillin-resistant Staphylococcus aureus (MRSA) is a World Health
Organization-listed priority pathogen. Scarce genomic data are available for MRSA
isolates from Malaysia. Here, we present the complete genome sequence of a
multidrug-resistant MRSA strain SauR3, isolated from the blood of a 6-year-old
patient hospitalized in Terengganu, Malaysia, in 2016. S. aureus SauR3 was
resistant to five antimicrobial classes comprising nine antibiotics. The genome was
sequenced on the Illumina and Oxford Nanopore platforms and hybrid assembly was
performed to obtain its complete genome sequence. The SauR3 genome consists of a
circular chromosome of 2,800,017 bp and three plasmids designated pSauR3-1 (42,928
bp), pSauR3-2 (3011 bp), and pSauR3-3 (2473 bp). SauR3 belongs to sequence type 573
(ST573), a rarely reported sequence type of the staphylococcal clonal complex 1
(CC1) lineage, and harbors a variant of the staphylococcal cassette chromosome mec
(SCCmec) type V (5C2&5) element which also contains the aac(6′)-
aph(2″) aminoglycoside-resistance genes. pSauR3-1 harbors several antibiotic
resistance genes in a 14,095 bp genomic island (GI), previously reported in the
chromosome of other staphylococci. pSauR3-2 is cryptic, whereas pSauR3-3 encodes
the ermC gene that mediates inducible resistance to macrolide-lincosamide-
streptogramin B (iMLSB). The SauR3 genome can potentially be used as a reference
genome for other ST573 isolates. Centre for Research in Infectious Diseases and
Biotechnology (CeRIDB), Faculty of Medicine, Universiti Sultan Zainal Abidin, Kuala
Terengganu 20400, Malaysia esratradat@gmail.com; ainalmardziah89@gmail.com;
suatmoi@um.edu.my; khchua@um.edu.my; muhammad.zarulhanifah@monash.edu;
qasim.ayub@monash.edu; prasit.pal@mahidol.ac.th; s.kwong@westernsydney.edu.au;
chewch@unisza.edu.my; chewchieng@gmail.com 10.3390/pathogens12030502 2023
12 3 - - 502 -
Singh, Swarn; Chauhan, Keerti; Bharadwaj, Atul; Kishore, Vimal; Laux, Peter; Luch,
Andreas; Singh, Ajay Polymer Translocation and Nanopore Sequencing: A Review of
Advances and Challenges International Journal of Molecular Sciences EN Review
polymer translocation; nanopore sequencing; translocation dynamics; nanopores
Various biological processes involve the translocation of macromolecules
across nanopores; these pores are basically protein channels embedded in membranes.
Understanding the mechanism of translocation is crucial to a range of technological
applications, including DNA sequencing, single molecule detection, and controlled
drug delivery. In this spirit, numerous efforts have been made to develop polymer
translocation-based sequencing devices, these efforts include findings and insights
from theoretical modeling, simulations, and experimental studies. As much as the
past and ongoing studies have added to the knowledge, the practical realization of
low-cost, high-throughput sequencing devices, however, has still not been realized.
There are challenges, the foremost of which is controlling the speed of
translocation at the single monomer level, which remain to be addressed in order to
use polymer translocation-based methods for sensing applications. In this article,
we review the recent studies aimed at developing control over the dynamics of
polymer translocation through nanopores. Department of Physics, Mahila
Mahavidyalaya (MMV), Banaras Hindu University, Varanasi 221005, UP, India
swarn@bhu.ac.in; keertichauhan1234@gmail.com; atulsbharadwaj@gmail.com;
vimalk@bhu.ac.in; peter.laux@bfr.bund.de; andreas.luch@bfr.bund.de; ajay-
vikram.singh@bfr.bund.de 10.3390/ijms24076153 2023 24 7 - -
6153 -
Pellegrini, Francesco; Buonavoglia, Alessio; Omar, Ahmed; Diakoudi, Georgia;
Lucente, Maria; Odigie, Amienwanlen; Sposato, Alessio; Augelli, Raffaella; Camero,
Michele; Decaro, Nicola; Elia, Gabriella; Bányai, Krisztián; Martella, Vito;
Lanave, Gianvito A Cold Case of Equine Influenza Disentangled with Nanopore
Sequencing Animals EN Article equine influence; sequencing techniques;
animal importation Massive sequencing techniques have allowed us to develop
straightforward approaches for the whole genome sequencing of viruses, including
influenza viruses, generating information that is useful for improving the levels
and dimensions of data analysis, even for archival samples. Using the Nanopore
platform, we determined the whole genome sequence of an H3N8 equine influenza
virus, identified from a 2005 outbreak in Apulia, Italy, whose origin had remained
epidemiologically unexplained. The virus was tightly related (>99% at the
nucleotide level) in all the genome segments to viruses identified in Poland in
2005–2008 and it was seemingly introduced locally with horse trading for the
meat industry. In the phylogenetic analysis based on the eight genome segments,
strain ITA/2005/horse/Bari was found to cluster with sub-lineage Florida 2 in the
HA and M genes, whilst in the other genes it clustered with strains of the Eurasian
lineage, revealing a multi-reassortant nature. Department of Veterinary Medicine,
University of Bari, 70010 Valenzano, Italy francesco.pellegrini@uniba.it;
alessio.buonavoglia85@gmail.com; ahmed.omar@uniba.it; georgia.diakoudi@uniba.it;
mariastella.lucente@uniba.it; amienwanlen.odigie@uniba.it;
alessio.sposato@izspb.it; r.augelli@sanita.it; michele.camero@uniba.it;
nicola.decaro@uniba.it; gabriella.elia@uniba.it; bkrota@hotmail.com;
vito.martella@uniba.it; gianvito.lanave@uniba.it 10.3390/ani13071153 2023
13 7 - - 1153 -
Bold, Dashzeveg; Souza-Neto, Jayme; Gombo-Ochir, Delgerzul; Gaudreault, Natasha;
Meekins, David; McDowell, Chester; Zayat, Batsukh; Richt, Juergen Rapid
Identification of ASFV, CSFV and FMDV from Mongolian Outbreaks with MinION Short
Amplicon Sequencing Pathogens EN Brief Report transboundary animal
diseases; African swine fever virus; classical swine fever virus; foot-and-mouth
disease virus; point-of-care diagnostics; MinION; short amplicon sequencing
African swine fever virus (ASFV), classical swine fever virus (CSFV), and
foot-and-mouth disease virus (FMDV) cause important transboundary animal diseases
(TADs) that have a significant economic impact. The rapid and unequivocal
identification of these pathogens and distinction from other animal diseases based
on clinical symptoms in the field is difficult. Nevertheless, early pathogen
detection is critical in limiting their spread and impact as is the availability of
a reliable, rapid, and cost-effective diagnostic test. The purpose of this study
was to evaluate the feasibility to identify ASFV, CSFV, and FMDV in field samples
using next generation sequencing of short PCR products as a point-of-care
diagnostic. We isolated nucleic acids from tissue samples of animals in Mongolia
that were infected with ASFV (2019), CSFV (2015), or FMDV (2018), and performed
conventional (RT-) PCR using primers recommended by the Terrestrial Animal Health
Code of the World Organization for Animal Health (WOAH). The (RT-) PCR products
were then sequenced in Mongolia using the MinION nanopore portable sequencer. The
resulting sequencing reads successfully identified the respective pathogens that
exhibited 91–100% nucleic acid similarity to the reference strains.
Phylogenetic analyses suggest that the Mongolian virus isolates are closely related
to other isolates circulating in the same geographic region. Based on our results,
sequencing short fragments derived by conventional (RT-) PCR is a reliable approach
for rapid point-of-care diagnostics for ASFV, CSFV, and FMDV even in low-resource
countries. Department of Diagnostic Medicine/Pathobiology, College of Veterinary
Medicine, Kansas State University, Manhattan, KS 66506, USAbold@vet.k-state.edu;
jsouzaneto@vet.k-state.edu; delgerzul@scvl.gov.mn; nng5757@vet.k-state.edu;
dmeekins@vet.k-state.edu; cdmcdow@vet.k-state.edu; zbatsukh@mail.mn; jricht@ksu.edu
10.3390/pathogens12040533 2023 12 4 - - 533 -
Kann, Simone; Concha, Gustavo; Weinreich, Felix; Hahn, Andreas; Rückert, Christian;
Kalinowski, Jörn; Landt, Olfert; Frickmann, Hagen Comparative Assessment of Two
Commercial Real-Time PCR Assays for the Diagnosis of Trypanosoma cruzi DNA in Serum
Microorganisms EN Article Chagas; diagnostic; molecular detection;
test comparison; Colombia; sensitivity; specificity This study was performed to
comparably assess two commercial real-time PCR assays for the identification of
Trypanosoma cruzi DNA in serum. A total of 518 Colombian serum samples with high
pre-test probability for infections with either T. cruzi or apathogenic Trypanosoma
rangeli were assessed. The assessment comprised the NDO real-time PCR (TIB MOLBIOL,
ref. no. 53-0755-96, referred to as the TibMolBiol assay in the following) with
specificity for T. cruzi and the RealStar Chagas PCR Kit 1.0 (altona DIAGNOSTICS,
order no. 611013, referred to as the RealStar assay in the following) targeting a
kinetoplast sequence of both T. cruzi and T. rangeli without further
discrimination. To discriminate between T. cruzi- and T. rangeli-specific real-time
PCR amplicons, Sanger sequencing results were available for a minority of cases
with discordant real-time PCR results, while the amplicons of the remaining
discordant samples were subjected to nanopore sequencing. The study assessment
indicated a proportion of 18.1% (n = 94) T. cruzi-positive samples next to 24
samples (4.6%) containing DNA of the phylogenetically related but apathogenic
parasite T. rangeli. The observed diagnostic accuracy as expressed by sensitivity
and specificity was 97.9% (92/94) and 99.3% (421/424) with the TibMolBiol assay and
96.8% (91/94) and 95.0% (403/424) with the RealStar assay, respectively. Reduced
specificity resulted from cross-reaction with T. rangeli in all instances (3 cross-
reactions with the TibMolBiol assay and 21 cross-reactions with the RealStar
assay). DNA from the six discrete typing units (DTUs) of T. cruzi was successfully
amplified by both real-time PCR assays. In summary, both assays showed a comparable
diagnostic accuracy for the diagnosis of T. cruzi from human serum, with a slightly
higher specificity seen for the TibMolBiol assay. The pronounced co-amplification
of DNA from apathogenic T. rangeli according to the RealStar assay may be a
disadvantage in areas of co-circulation with T. cruzi, while the test performance
of the two compared assays will be quite similar in geographic settings where T.
rangeli infections are unlikely. Medical Mission Institute, 97074 Würzburg,
Germany simone_kann@hotmail.com; gustavoconcha16@gmail.com;
felixweinreich@bundeswehr.org; andreas.hahn@uni-rostock.de;
christian.rueckert@cebitec.uni-bielefeld.de; joern@cebitec.uni-bielefeld.de;
olandt@tib-molbiol.de; frickmann@bnitm.de 10.3390/microorganisms11040901 2023
11 4 - - 901 -
Lee, Dong-Jun; Choi, Ji-Weon; Kang, Ji-Nam; Lee, Si-Myung; Park, Gyu-Hwang; Kim,
Chang-Kug Chromosome-Scale Genome Assembly and Triterpenoid Saponin Biosynthesis
in Korean Bellflower (Platycodon grandiflorum) International Journal of Molecular
Sciences EN Brief Report chromosome-scale genome; Platycodon
grandiflorum; triterpenoid saponin biosynthesis Platycodon grandiflorum belongs to
the Campanulaceae family and is an important medicinal and food plant in East Asia.
However, on the whole, the genome evolution of P. grandiflorum and the molecular
basis of its major biochemical pathways are poorly understood. We reported a
chromosome-scale genome assembly of P. grandiflorum based on a hybrid method using
Oxford Nanopore Technologies, Illumina sequences, and high-throughput chromosome
conformation capture (Hi-C) analysis. The assembled genome was finalized as 574 Mb,
containing 41,355 protein-coding genes, and the genome completeness was assessed as
97.6% using a Benchmarking Universal Single-Copy Orthologs analysis. The P.
grandiflorum genome comprises nine pseudo-chromosomes with 56.9% repeat sequences,
and the transcriptome analysis revealed an expansion of the 14 beta-amylin genes
related to triterpenoid saponin biosynthesis. Our findings provide an understanding
of P. grandiflorum genome evolution and enable genomic-assisted breeding for the
mass production of important components such as triterpenoid saponins. Genomics
Division, National Institute of Agricultural Sciences, Jeonju 54874, Republic of
Korea leemoses1004@gmail.com; jwcnpri@korea.kr; greatnami@korea.kr;
tataby@korea.kr; guhwang01@korea.kr; chang@korea.kr 10.3390/ijms24076534 2023
24 7 - - 6534 -
Ren, Wangmei; Wang, Liying; Feng, Guangcheng; Tao, Cheng; Liu, Yongsheng; Yang, Jun
High-Quality Assembly and Comparative Analysis of Actinidia latifolia and A.
valvata Mitogenomes Genes EN Article Actinidia latifolia; Actinidia
valvata; mitogenome; phylogenetic analysis Kiwifruit (Actinidia) has been
recently domesticated as a horticultural crop with remarkably economic and
nutritional value. In this study, by combining sequence datasets from Oxford
Nanopore long-reads and Illumina short-reads, we de novo assembled two mitogenomes
of Actinidia latifolia and A. valvata, respectively. The results indicated that the
A. latifolia mitogenome has a single, circular, 825,163 bp molecule while the A.
valvata mitogenome possesses two distinct circular molecules, 781,709 and 301,558
bp, respectively. We characterized the genome structure, repeated sequences, DNA
transfers, and dN/dS selections. The phylogenetic analyses showed that A. valvata
and A. arguta, or A. latifolia and A. eriantha, were clustered together,
respectively. This study provides valuable sequence resources for evolutionary
study and molecular breeding in kiwifruit. College of Horticulture, Anhui
Agriculture University, Hefei 350002, China 326569730@sina.com;
472550880@163.com; 1208720430@sina.com; 1592437499@163.com;
liuyongsheng1122@ahau.edu.cn; yj1904735520@sina.com 10.3390/genes14040863 2023
14 4 - - 863 -
Hernandez, Miguel; Hernandez, Gabriela; Portillo, Roberto; Rubio, Efraín;
Petranovskii, Vitalii; Alvarez, Karin; Velasco, Ma; Santamaría, Juana; Tornero,
Mario; Paniagua, Laura CO2 Adsorption on Natural Zeolites from Puebla,
México, by Inverse Gas Chromatography Separations EN Article
nanoporosity; CO2; adsorption; clinoptilolite; inverse chromatography The
applicability of clinoptilolite zeolites in controlling the emission of greenhouse
gases (GHGs) such as CO2, the most significant GHG, is investigated herein. In this
research, Mexican natural zeolites (ATN) originating from an Atzinco deposit in the
state of Puebla were used. Samples of modified clinoptilolite (ATH4, ATH3, ATH2 and
ATH1) were obtained from the starting material by acid treatment of various
intensities. Inverse gas chromatography was used to evaluate CO2 adsorption in
clinoptilolite, natural and chemically modified. Adsorption of CO2 was investigated
in the temperature range of 433–573 K, using a TCD detector, and He as a
carrier gas. The experimental CO2 adsorption data were processed by Freundlich and
Langmuir equations. The degree of interaction between CO2 and the dealuminated
clinoptilolite samples was examined through the evaluation of the isosteric
enthalpy of adsorption. This calculation was made by using the
Clausius–Clapeyron equation, which established the following sequence: ATH1
> ATH2 > ATH4 > ATN > ATH3. The nanoporosity of these clinoptolite
zeolites from new deposit in sedimentary rocks was studied through HRADS adsorption
of N2. Simultaneously, these zeolites were, respectively, characterized by XRD,
EDS, and SEM. Micropores are described by the Dubinin–Asthakov distribution.
Various adsorption mechanisms that occur in these nanoporous materials at different
relative pressures can be visualized. The quantitative determination of starting
mineral is described as: Ca-Clinoptilolite (88.76%) >> Montmorillonite
(11.11%) >> quartz (0.13%). The Si/Al molar ratio after acid treatment is:
ATH4 > ATH2 > ATN > ATH3 > ATH1. The Langmuir specific surface area
(ASL) varies as follows: ATN > ATH2 > ATH4 > ATH3 > ATH1. At the same
time, the VΣ values are as follows: ATN > ATH4 > ATH3 > ATH1 >
ATH2. Departament of Zeolites Research, Postgraduate in Agroecology, ICUAP,
Meritorious Autonomous University of Puebla, Puebla City 72570, Mexico
miguel.hernandez@correo.buap.mx; g.hernandez@gmail.com;
roberto.portillo@correo.buap.mx; efrain.rubio@correo.buap.mx; vitalii@cnyn.unam.mx;
wiki.ecko@gmail.com; angeles.velasco@correo.buap.mx;
deisy.santamaria@correo.buap.mx; mario.tornero@correo.buap.mx;
laura.paniagua@correo.buap.mx 10.3390/separations10040238 2023 10 4 -
- 238 -
Wang, Ziyi; Du, Yujiao; Li, Suhao; Xu, Xuewen; Chen, XuehaoA Complete Genome
Sequence of Podosphaera xanthii Isolate YZU573, the Causal Agent of Powdery Mildew
Isolated from Cucumber in China Pathogens EN Communication Podosphaera
xanthii; YZU573; cucumber; genome assembly Podosphaera xanthii is a well-known
obligate biotrophic pathogen that causes powdery mildew (PM) disease on
cucurbitaceous plants and is one of the most important limiting factors for
cucumber production worldwide. To better understand the avirulence effector
proteins in this species that are known to be involved in host-pathogen
interaction, the draft genome assembly of P. xanthii isolate YZU573 from cucumber
leaves with symptoms of PM was obtained with a hybrid approach, combining nanopore
long-read and llumina paired-end sequencing. The final P. xanthii YZU573 genome
assembly of 152.7 Mb consists of 58 contigs, with an N50 value of 0.75 Mb and 6491
predicted protein-coding genes. The effector analysis using the whole-genome
sequence information revealed a total of 87 putative effector candidates, and 65 of
them had their analogs, whereas the remaining 22 were novel ones. The new P.
xanthii genome provides valuable resources to better understand plant-microbe
interaction in cucumber PM disease. School of Horticulture and Landscape
Architecture, Yangzhou University, Yangzhou 225009, China wrince73@163.com;
dd18752595989@163.com; hli808863@gmail.com; xxu323@yzu.edu.cn; xhchen@yzu.edu.cn
10.3390/pathogens12040561 2023 12 4 - - 561 -
Laidoudi, Younes; Rousset, Elodie; Dessimoulie, Anne-Sophie; Prigent, Myriam;
Raptopoulo, Alizée; Huteau, Quentin; Chabbert, Elisabeth; Navarro, Catherine;
Fournier, Pierre-Edouard; Davoust, Bernard Tracking the Source of Human Q
Fever from a Southern French Village: Sentinel Animals and Environmental Reservoir
Microorganisms EN Article One Health; Coxiella burnetii; Q fever;
sentinels; epidemiology Coxiella burnetii, also known as the causal agent of Q
fever, is a zoonotic pathogen infecting humans and several animal species. Here, we
investigated the epidemiological context of C. burnetii from an area in the
Hérault department in southern France, using the One Health paradigm. In
total, 13 human cases of Q fever were diagnosed over the last three years in an
area comprising four villages. Serological and molecular investigations conducted
on the representative animal population, as well as wind data, indicated that some
of the recent cases are likely to have originated from a sheepfold, which revealed
bacterial contamination and a seroprevalence of 47.6%. However, the clear-cut
origin of human cases cannot be ruled out in the absence of molecular data from the
patients. Multi-spacer typing based on dual barcoding nanopore sequencing
highlighted the occurrence of a new genotype of C. burnetii. In addition, the
environmental contamination appeared to be widespread across a perimeter of 6 km
due to local wind activity, according to the seroprevalence detected in dogs
(12.6%) and horses (8.49%) in the surrounding populations. These findings were
helpful in describing the extent of the exposed area and thus supporting the use of
dogs and horses as valuable sentinel indicators for monitoring Q fever. The present
data clearly highlighted that the epidemiological surveillance of Q fever should be
reinforced and improved. Aix Marseille University, IRD, AP-HM, MEPHI, 13005
Marseille, France younes.laidoudi@yahoo.com; elodie.rousset@anses.fr;
cliniquedes4chemins@gmail.com; myriam.prigent@anses.fr; alizee.raptopoulo@anses.fr;
huteau.quentin@lesmandailles.fr; elisabeth.chabbert@biomed34.fr;
laurencep34@gmail.com; pierre-edouard.fournier@univ-amu.fr;
bernard.davoust@gmail.com 10.3390/microorganisms11041016 2023 11 4
- - 1016 -
Zhao, Chunhua; Feng, Xi-long; Wang, Zhen-xin; Qi, Jianzhao The First Whole Genome
Sequencing of Agaricus bitorquis and Its Metabolite Profiling Journal of Fungi
EN Article Agaricus bitorquis; comparative genome; mitogenome; edible
mushroom Agaricus bitorquis, an emerging wild mushroom with remarkable
biological activities and a distinctive oversized mushroom shape, has gained
increasing attention in recent years. Despite its status as an important resource
of wild edible fungi, knowledge about this mushroom is still limited. In this
study, we used the Illumina NovaSeq and Nanopore PromethION platforms to sequence,
de novo assemble, and annotate the whole genome and mitochondrial genome
(mitogenome) of the A. bitorquis strain BH01 isolated from Bosten Lake, Xinjiang
Province, China. Using the genome-based biological information, we identified
candidate genes associated with mating type and carbohydrate-active enzymes in A.
bitorquis. Cluster analysis based on P450 of basidiomycetes revealed the types of
P450 members of A. bitorquis. Comparative genomic, mitogenomic, and phylogenetic
analyses were also performed, revealing interspecific differences and evolutionary
features of A. bitorquis and A. bisporus. In addition, the molecular network of
metabolites was investigated, highlighting differences in the chemical composition
and content of the fruiting bodies of A. bitorquis and A. bisporus. The genome
sequencing provides a comprehensive understanding and knowledge of A. bitorquis and
the genus Agaricus mushrooms. This work provides valuable insights into the
potential for artificial cultivation and molecular breeding of A. bitorquis, which
will facilitate the development of A. bitorquis in the field of edible mushrooms
and functional food manufacture. Hubei Key Laboratory of Natural Medicinal
Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College,
Huazhong University of Science and Technology, Wuhan 430030, China
zhaochunhua@whu.edu.cn; elf-nar@nwafu.edu.cn; 3346462730@nwafu.edu.cn;
qjz@nwafu.edu.cn 10.3390/jof9040485 2023 9 4 - - 485 -
Garcia-Segura, Sergio; del Rey, Javier; Closa, Laia; Garcia-Martínez, Iris;
Hobeich, Carlos; Castel, Ana; Vidal, Francisco; Benet, Jordi; Oliver-Bonet, Maria
Characterization of Seminal Microbiome of Infertile Idiopathic Patients Using
Third-Generation Sequencing Platform International Journal of Molecular
Sciences EN Article seminal microbiota; MinION; nanopore sequencing;
Illumina; male fertility Since the first description of a commensal seminal
microbiome using sequencing, less than a decade ago, interest in the composition of
this microbiome and its relationship with fertility has been growing. Articles
using next-generation sequencing techniques agree on the identification of the most
abundant bacterial phyla. However, at the genus level, there is still no consensus
on which bacteria are most abundant in human seminal plasma. This discrepancy may
be due to methodological variability such as sample collection, bacterial DNA
extraction methodology, which hypervariable regions of 16S rRNA gene have been
amplified, or bioinformatic analysis. In the present work, seminal microbiota of 14
control samples and 42 samples of idiopathic infertile patients were characterized
based on full-length sequencing of the 16S rRNA gene using MinION platform from
Oxford Nanopore. These same samples had been analyzed previously using
Illumina’s MiSeq sequencing platform. Comparison between the results obtained
with the two platforms has been used to analyze the impact of sequencing method on
the study of the seminal microbiome’s composition. Seminal microbiota
observed with MinION were mainly composed of the phyla Firmicutes, Proteobacteria,
Bacteroidetes and Actinobacteria, with the most abundant genera being
Peptoniphilus, Finegoldia, Staphylococcus, Anaerococcus, Campylobacter, Prevotella,
Streptococcus, Lactobacillus, Ezakiella and Enterococcus. This composition was
similar to that found by the Illumina platform, since these 10 most abundant genera
were also among the most abundant genera detected by the Nanopore platform. In both
cases, the top 10 genera represented more than 70% of the classified reads.
However, relative abundance of each bacterium did not correlate between these two
platforms, with intraindividual variations of up to 50 percentage points in some
cases. Results suggest that the effect of the sequencing platform on the
characterization of seminal microbiota is not very large at the phylum level, with
slightly variances in Firmicutes and Actinobacteria, but presents differences at
the genus level. These differences could alter the composition and diversity of
bacterial profiles or posterior analyses. This indicates the importance of
conducting multi-platform studies to better characterize seminal microbioma. Unit
of Cell Biology and Medical Genetics, Department of Cell Biology, Physiology and
Immunology, Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Spain
sergio.garcia.segura@uab.cat; javier.delrey@uab.cat; lclosa@bst.cat;
igarcia@bst.cat; chobeich@bst.cat; abcastel@yahoo.es; fvidal@bst.cat;
jordi.benet@uab.cat; maria.oliver@uab.cat 10.3390/ijms24097867 2023 24 9
- - 7867 -
Herskind, Christinna; Petersen, Heidi; Pertoldi, Cino; Østergaard, Stine;
Kołodziej-Sobocińska, Marta; Sobociński, Wojciech; Tokarska, Małgorzata; Hammer
Jensen, Trine Effect of Translocation on Host Diet and Parasite Egg Burden: A
Study of the European Bison (Bison bonasus) Biology EN Article
European bison; eDNA; parasites; diet; strongyles; Lille Vildmose; Białowieża
Forest; EPG; Baermann; flotation; nanopore sequencing; rewilding; conservation
For the purpose of nature management and species conservation, European bison
(Bison bonasus) are being increasingly reintroduced into nature reserves across
Europe. The aim of this study was to investigate European bison’s
adaptability to new areas through the study of their parasite-EPG (eggs per gram
feces) and dietary diversity during twelve months after translocation. We compared
the parasite-EPG from introduced European bison in Lille Vildmose, Denmark, with
the parasite-EPG from populations from Bornholm, Denmark, and Białowieża
Forest, Poland. From March 2021 to February 2022, fecal samples were collected from
three populations. Samples from Lille Vildmose were examined through flotation,
sedimentation, the Baermann technique, and nanopore sequencing. Fecal samples from
Bornholm and Białowieża were examined through flotation and
sedimentation. Nanopore sequencing of DNA from 63 European bison’s fecal
samples collected during March–September in Lille Vildmose identified 8
species of nematodes within the digestive tract of the European bison, with
Haemonchus contortus being the most frequently observed. In Lille Vildmose, a
significantly higher excretion of nematode-EPG was observed during the summer
period than in the spring, autumn, and winter. In addition, monthly differences in
the excretion of nematode eggs were found, with this being significantly higher in
June than in the months during autumn and winter (October–February).
Significant differences in the nematode-EPG were only found between the excretion
of nematode eggs in Białowieża Forest when compared to that of Lille
Vildmose, with significantly higher excretion in Lille Vildmose
(October–November). The results indicate that the development rates for
nematodes may be affected by changes in temperature, with increasing temperatures
speeding up their development time. Independent of this study design, wildlife vets
together with the gamekeepers managing the herd found it necessary to treat the
herd with antiparasitics for practical and animal welfare reasons in relation to
translocation. Furthermore, 79 plant taxa were identified in the diet of the
European bison. The broadest diet was observed in March suggesting that the
European bison quickly adapted to their new habitat. The results suggest a seasonal
shift in their diet, with this being most apparent from March to April.
Department of Chemistry and Bioscience, Aalborg University, Fredrik Bajers
Vej 7H, 9220 Aalborg, Denmark cherskind@gmail.com; hepet@fvst.dk; cp@bio.aau.dk;
skoe@bio.aau.dk; mksobocinska@ibs.bialowieza.pl; w.sobocinski@uwb.edu.pl;
tokarska@ibs.bialowieza.pl; trine@bio.aau.dk 10.3390/biology12050680 2023 12
5 - - 680 -
Zhang, Yue; Zhang, Qian; Yang, Xingyu; Gu, Xiaofeng; Chen, Jinming; Shi, Tao 6mA
DNA Methylation on Genes in Plants Is Associated with Gene Complexity, Expression
and Duplication Plants EN Article N6-methyladenine; gene duplication;
gene expression; Nelumbo nucifera N6-methyladenine (6mA) DNA methylation has
emerged as an important epigenetic modification in eukaryotes. Nevertheless, the
evolution of the 6mA methylation of homologous genes after species and after gene
duplications remains unclear in plants. To understand the evolution of 6mA
methylation, we detected the genome-wide 6mA methylation patterns of four lotus
plants (Nelumbo nucifera) from different geographic origins by nanopore sequencing
and compared them to patterns in Arabidopsis and rice. Within lotus, the genomic
distributions of 6mA sites are different from the widely studied 5mC methylation
sites. Consistently, in lotus, Arabidopsis and rice, 6mA sites are enriched around
transcriptional start sites, positively correlated with gene expression levels, and
preferentially retained in highly and broadly expressed orthologs with longer gene
lengths and more exons. Among different duplicate genes, 6mA methylation is
significantly more enriched and conserved in whole-genome duplicates than in local
duplicates. Overall, our study reveals the convergent patterns of 6mA methylation
evolution based on both lineage and duplicate gene divergence, which underpin their
potential role in gene regulatory evolution in plants. CAS Key Laboratory of
Aquatic Botany and Watershed Ecology, Wuhan Botanical Garden, Chinese Academy of
Sciences, Wuhan 430074, China zhangyue@wbgcas.cn; zq_9020@163.com;
sanmu109@163.com; guxiaofeng@caas.cn; jmchen@wbgcas.cn; shitao323@wbgcas.cn
10.3390/plants12101949 2023 12 10 - - 1949 -
Di Profio, Federica; Sarchese, Vittorio; Fruci, Paola; Aste, Giovanni; Martella,
Vito; Palombieri, Andrea; Di Martino, Barbara Exploring the Enteric Virome of
Cats with Acute Gastroenteritis Veterinary Sciences EN Article
gastroenteritis; cats; viruses; PCRs and RT-PCRs; ONT sequencing Viruses are
a major cause of acute gastroenteritis (AGE) in cats, chiefly in younger animals.
Enteric specimens collected from 29 cats with acute enteritis and 33 non-diarrhoeic
cats were screened in PCRs and reverse transcription (RT) PCR for a large panel of
enteric viruses, including also orphan viruses of recent identification. At least
one viral species, including feline panleukopenia virus (FPV), feline enteric
coronavirus (FCoV), feline chaphamaparvovirus, calicivirus (vesivirus and
novovirus), feline kobuvirus, feline sakobuvirus A and Lyon IARC polyomaviruses,
was detected in 66.1% of the samples.. Co-infections were mainly accounted for by
FPV and FCoV and were detected in 24.2% of the samples. The virome composition was
further assessed in eight diarrhoeic samples, through the construction of
sequencing libraries using a sequence-independent single-primer amplification
(SISPA) protocol. The libraries were sequenced on Oxford Nanopore Technologies
sequencing platform. A total of 41 contigs (>100 nt) were detected from seven
viral families infecting mammals, included Parvoviridae, Caliciviridae,
Picornaviridae, Polyomaviridae, Anelloviridae, Papillomaviridae and
Paramyxoviridae, revealing a broad variety in the composition of the feline enteric
virome. Department of Veterinary Medicine, Università degli Studi di Teramo,
64100 Teramo, Italy fdiprofio@unite.it; vsarchese@unite.it; pfruci@unite.it;
gaste@unite.it; vito.martella@uniba.it; apalombieri@unite.it; bdimartino@unite.it
10.3390/vetsci10050362 2023 10 5 - - 362 -
Ciobanu, Cristian-Gabriel; Nucă, Irina; Popescu, Roxana; Antoci, Lucian-Mihai;
Caba, Lavinia; Ivanov, Anca; Cojocaru, Karina-Alexandra; Rusu, Cristina; Mihai,
Cosmin-Teodor; Pânzaru, Monica-Cristina Narrative Review: Update on the Molecular
Diagnosis of Fragile X Syndrome International Journal of Molecular Sciences
EN Review fragile X syndrome; long read; methylation; PacBio
sequencing; Oxford Nanoporesequencing The diagnosis and management of fragile X
syndrome (FXS) have significantly improved in the last three decades, although the
current diagnostic techniques are not yet able to precisely identify the number of
repeats, methylation status, level of mosaicism, and/or the presence of AGG
interruptions. A high number of repeats (>200) in the fragile X messenger
ribonucleoprotein 1 gene (FMR1) results in hypermethylation of promoter and gene
silencing. The actual molecular diagnosis is performed using a Southern blot, TP-
PCR (Triplet-Repeat PCR), MS-PCR (Methylation-Specific PCR), and MS-MLPA
(Methylation-Specific MLPA) with some limitations, with multiple assays being
necessary to completely characterise a patient with FXS. The actual gold standard
diagnosis uses Southern blot; however, it cannot accurately characterise all cases.
Optical genome mapping is a new technology that has also been developed to approach
the diagnosis of fragile X syndrome. Long-range sequencing represented by PacBio
and Oxford Nanopore has the potential to replace the actual diagnosis and offers a
complete characterization of molecular profiles in a single test. The new
technologies have improved the diagnosis of fragile X syndrome and revealed unknown
aberrations, but they are a long way from being used routinely in clinical
practice. Medical Genetics Department, Faculty of Medicine, “Grigore T. Popa”
University of Medicine and Pharmacy, University Street No 16, 700115 Iasi, Romania
ciobanucristian@yahoo.com; irina.resmerita@umfiasi.ro;
roxana.popescu2014@gmail.com; lucian-mihai.antoci@email.umfiasi.ro;
lavinia.caba@umfiasi.ro; anca_vi@yahoo.com; karina-alexandra.cojocaru@d.umfiasi.ro;
abcrusu@gmail.com; cosmin-teodor.mihai@laboratorpraxis.ro;
monica.panzaru@umfiasi.ro 10.3390/ijms24119206 2023 24 11 - -
9206 -
de Moraes, Laise; Portilho, Moyra; Vrancken, Bram; Van den Broeck, Frederik;
Santos, Luciane; Cucco, Marina; Tauro, Laura; Kikuti, Mariana; Silva, Monaise;
Campos, Gúbio; Reis, Mitermayer; Barral, Aldina; Barral-Netto, Manoel; Boaventura,
Viviane; Vandamme, Anne-Mieke; Theys, Kristof; Lemey, Philippe; Ribeiro, Guilherme;
Khouri, Ricardo Analyses of Early ZIKV Genomes Are Consistent with Viral Spread
from Northeast Brazil to the Americas Viruses EN Communication Zika;
arboviruses; vector-borne infections; genomic surveillance; phylogenetics The
Americas, particularly Brazil, were greatly impacted by the widespread Zika virus
(ZIKV) outbreak in 2015 and 2016. Efforts were made to implement genomic
surveillance of ZIKV as part of the public health responses. The accuracy of
spatiotemporal reconstructions of the epidemic spread relies on the unbiased
sampling of the transmission process. In the early stages of the outbreak, we
recruited patients exhibiting clinical symptoms of arbovirus-like infection from
Salvador and Campo Formoso, Bahia, in Northeast Brazil. Between May 2015 and June
2016, we identified 21 cases of acute ZIKV infection and subsequently recovered 14
near full-length sequences using the amplicon tiling multiplex approach with
nanopore sequencing. We performed a time-calibrated discrete phylogeographic
analysis to trace the spread and migration history of the ZIKV. Our phylogenetic
analysis supports a consistent relationship between ZIKV migration from Northeast
to Southeast Brazil and its subsequent dissemination beyond Brazil. Additionally,
our analysis provides insights into the migration of ZIKV from Brazil to Haiti and
the role Brazil played in the spread of ZIKV to other countries, such as Singapore,
the USA, and the Dominican Republic. The data generated by this study enhances our
understanding of ZIKV dynamics and supports the existing knowledge, which can aid
in future surveillance efforts against the virus. Programa de Pós-Graduação em
Ciências da Saúde, Faculdade de Medicina da Bahia, Universidade Federal da Bahia,
Salvador 40026-010, Brazil laise.moraes@fiocruz.br; moyra.portilho@fiocruz.br;
bram.vrancken@ulb.be; frederik.vandenbroeck@uantwerpen.be;
lucianeamorim@bahiana.edu.br; marina.cucco@fiocruz.br; lauratauro@gmail.com;
marianakikuti@gmail.com; monaise.sc@gmail.com; gubio@ufba.br;
mitermayer.reis@fiocruz.br; aldina.barral@fiocruz.br; manoel.barral@fiocruz.br;
viviane.boaventura@fiocruz.br; annemie.vandamme@kuleuven.be;
kristof.theys@kuleuven.be; philippe.lemey@kuleuven.be;
guilherme.ribeiro@fiocruz.br; ricardo.khouri@fiocruz.br 10.3390/v15061236 2023
15 6 - - 1236 -
Bologa, Alexandru; Stoica, Ileana; Constantin, Nicoleta; Ecovoiu, Alexandru The
Landscape of the DNA Transposons in the Genome of the Horezu_LaPeri Strain of
Drosophila melanogaster Insects EN Article transposable elements; DNA
natural transposons; Drosophila melanogaster; P-element; heterochromatin;
bioinformatics; transposon annotation Natural transposons (NTs) represent
mobile DNA sequences found in both prokaryotic and eukaryotic genomes. Drosophila
melanogaster (the fruit fly) is a eukaryotic model organism with NTs standing for
about 20% of its genome and has contributed significantly to the understanding of
various aspects of transposon biology. Our study describes an accurate approach
designed to map class II transposons (DNA transposons) in the genome of the
Horezu_LaPeri fruit fly strain, consecutive to Oxford Nanopore Technology
sequencing. A whole genome bioinformatics analysis was conducted using Genome
ARTIST_v2, LoRTE and RepeatMasker tools to identify DNA transposons insertions.
Then, a gene ontology enrichment analysis was performed in order to evaluate the
potential adaptive role of some DNA transposons insertions. Herein, we describe DNA
transposon insertions specific for the Horezu_LaPeri genome and a predictive
functional analysis of some insertional alleles. The PCR validation of P-element
insertions specific for this fruit fly strain, along with a putative consensus
sequence for the KP element, is also reported. Overall, the genome of the
Horezu_LaPeri strain contains several insertions of DNA transposons associated with
genes known to be involved in adaptive processes. For some of these genes,
insertional alleles obtained via mobilization of the artificial transposons were
previously reported. This is a very alluring aspect, as it suggests that
insertional mutagenesis experiments conducting adaptive predictions for laboratory
strains may be confirmed by mirroring insertions which are expected to be found at
least in some natural fruit fly strains. Department of Genetics, Faculty of
Biology, University of Bucharest, 060101 Bucharest, Romania
alexandru.bologa@drd.unibuc.ro; ileana.stoica@bio.unibuc.ro;
constantin.nicoleta-denisa@s.bio.unibuc.ro; alexandru.ecovoiu@bio.unibuc.ro
10.3390/insects14060494 2023 14 6 - - 494 -
Li, Kathy; Lau, Betty; Suárez, Nicolás; Camiolo, Salvatore; Gunson, Rory; Davison,
Andrew; Orton, Richard Direct Nanopore Sequencing of Human Cytomegalovirus Genomes
from High-Viral-Load Clinical Samples Viruses EN Article human
cytomegalovirus; clinical sample; genome; nanopore sequencing; Illumina sequencing
Nanopore sequencing is becoming increasingly commonplace in clinical
settings, particularly for diagnostic assessments and outbreak investigations, due
to its portability, low cost, and ability to operate in near real-time. Although
high sequencing error rates initially hampered the wider implementation of this
technology, improvements have been made continually with each iteration of the
sequencing hardware and base-calling software. Here, we assess the feasibility of
using nanopore sequencing to determine the complete genomes of human
cytomegalovirus (HCMV) in high-viral-load clinical samples without viral DNA
enrichment, PCR amplification, or prior knowledge of the sequences. We utilised a
hybrid bioinformatic approach that involved assembling the reads de novo, improving
the consensus sequence by aligning reads to the best-matching genome from a
collated set of published sequences, and polishing the improved consensus sequence.
The final genomes from a urine sample and a lung sample, the former with an HCMV to
human DNA load approximately 50 times greater than the latter, achieved 99.97 and
99.93% identity, respectively, to the benchmark genomes obtained independently by
Illumina sequencing. Thus, we demonstrated that nanopore sequencing is capable of
determining HCMV genomes directly from high-viral-load clinical samples with a high
accuracy. Medical Research Council, University of Glasgow Centre for Virus
Research, Glasgow G61 1QH, UK kathy.li@glasgow.ac.uk; betty-lau@hotmail.com;
nicolas.suarez@glasgow.ac.uk; salvocamiolo@gmail.com; rory.gunson@ggc.scot.nhs.uk;
andrew.davison@glasgow.ac.uk; richard.orton@glasgow.ac.uk 10.3390/v15061248 2023
15 6 - - 1248 -
Baltazar, Elsa; Rodrigues, Sara; Ares, Aitana; Camelo, Alexandra; Brandão, Inês;
Espirito Santo, Christophe; Trovão, João; Garcia, Eva; Costa, Joana
Morphological, Molecular and Genomic Identification and Characterisation of
Monilinia fructicola in Prunus persica from Portugal Agronomy EN Article
brown rot; Cova da Beira; first report; whole genome sequence; pathogenicity
tests In Portugal, the Cova da Beira region is well-known for the production of
Prunus spp. and is considered the main peach production area in the country. In the
spring of 2021 and 2022, field surveys in peach and nectarine orchards showed
symptoms of decline such as cankers, gummosis, dry branches, abortion of flowers,
mummified fruits and the partial or total death of some plants. Brown rot is caused
by three species of the genus Monilinia, M. fructigena, M. laxa and M. fructicola,
the last is an OEPP/EPPO A2 quarantine organism on peach trees. Brown rot disease
had previously been described in the Cova da Beira region, however, the recent high
mortality and severity of symptoms raised doubts as to the species involved.
Symptomatic plant material was collected from thirteen orchards and used for fungal
isolation and molecular detection according to the OEPP/EPPO standard. M.
fructicola was confirmed morphologically and molecularly in two orchards, and
molecularly (duplex real-time PCR) detected in two others. Whole genome sequencing
using Oxford Nanopore MinION was also carried out to confirm the identification.
Pathogenicity tests were performed on peach, nectarine and sweet cherry fruit
according to Koch’s postulates. Based on all the results obtained, we report
the first detection of M. fructicola in P. persica in Portugal. University of
Coimbra - Center for Functional Ecology Science for People & the Planet, TERRA
Associated Laboratory, Department of Life Sciences, Calçada Martim de Freitas,
Coimbra 3000-456, Portugal e.c.s.baltazar@gmail.com; srodrigues@ipn.pt;
bioaitana26@gmail.com; alexandra.camelo@cataa.pt; inesbrandao@cataa.pt;
cespiritosanto@cataa.pt; jtrovao@ipn.pt; egarcia@ipn.pt; jcosta@uc.pt
10.3390/agronomy13061493 2023 13 6 - - 1493 -
Merkulov, Pavel; Egorova, Ekaterina; Kirov, Ilya Composition and Structure of
Arabidopsis thaliana Extrachromosomal Circular DNAs Revealed by Nanopore Sequencing
Plants EN Communication extrachromosomal circular DNAs;
Arabidopsis; long-read sequencing; transposable elements Extrachromosomal
circular DNAs (eccDNAs) are enigmatic DNA molecules that have been detected in a
range of organisms. In plants, eccDNAs have various genomic origins and may be
derived from transposable elements. The structures of individual eccDNA molecules
and their dynamics in response to stress are poorly understood. In this study, we
showed that nanopore sequencing is a useful tool for the detection and structural
analysis of eccDNA molecules. Applying nanopore sequencing to the eccDNA molecules
of epigenetically stressed Arabidopsis plants grown under various stress treatments
(heat, abscisic acid, and flagellin), we showed that TE-derived eccDNA quantity and
structure vary dramatically between individual TEs. Epigenetic stress alone did not
cause eccDNA up-regulation, whereas its combination with heat stress triggered the
generation of full-length and various truncated eccDNAs of the ONSEN element. We
showed that the ratio between full-length and truncated eccDNAs is TE- and
condition-dependent. Our work paves the way for further elucidation of the
structural features of eccDNAs and their connections with various biological
processes, such as eccDNA transcription and eccDNA-mediated TE silencing. Moscow
Institute of Physics and Technology, 141701 Dolgoprudny, Russia
paulmerkulov97@gmail.com; egorova.ekaterina20021120@gmail.com;
kirovez@gmail.com 10.3390/plants12112178 2023 12 11 - - 2178 -
Liu, Miao; Li, Junyang; Tan, Cherie Unlocking the Power of Nanopores: Recent
Advances in Biosensing Applications and Analog Front-End Biosensors EN Review
nanopores; biosensors; DNA sequencing; protein sequencing; chiral molecules;
transimpedance amplifiers The biomedical field has always fostered innovation
and the development of various new technologies. Beginning in the last century,
demand for picoampere-level current detection in biomedicine has increased, leading
to continuous breakthroughs in biosensor technology. Among emerging biomedical
sensing technologies, nanopore sensing has shown great potential. This paper
reviews nanopore sensing applications, such as chiral molecules, DNA sequencing,
and protein sequencing. However, the ionic current for different molecules differs
significantly, and the detection bandwidths vary as well. Therefore, this article
focuses on current sensing circuits, and introduces the latest design schemes and
circuit structures of different feedback components of transimpedance amplifiers
mainly used in nanopore DNA sequencing. Medical College, Tianjin University,
Tianjin 300072, China 3005202010@tju.edu.cn; ljyzy@tju.edu.cn;
cherie.tan@tju.edu.cn 10.3390/bios13060598 2023 13 6 - - 598
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Zhang, Zhihao; Xia, Tian; Zhou, Shengyang; Yang, Xiufeng; Lyu, Tianshu; Wang,
Lidong; Fang, Jiaohui; Wang, Qi; Dou, Huashan; Zhang, Honghai High-Quality
Chromosome-Level Genome Assembly of the Corsac Fox (Vulpes corsac) Reveals
Adaptation to Semiarid and Harsh Environments International Journal of Molecular
Sciences EN Article chromosome-level genome; Hi-C; semiarid adaption;
Vulpes corsac; diet strategy The Corsac fox (Vulpes corsac) is a species of fox
distributed in the arid prairie regions of Central and Northern Asia, with distinct
adaptations to dry environments. Here, we applied Oxford-Nanopore sequencing and a
chromosome structure capture technique to assemble the first Corsac fox genome,
which was then assembled into chromosome fragments. The genome assembly has a total
length of 2.2 Gb with a contig N50 of 41.62 Mb and a scaffold N50 of 132.2 Mb over
18 pseudo-chromosomal scaffolds. The genome contained approximately 32.67% of
repeat sequences. A total of 20,511 protein-coding genes were predicted, of which
88.9% were functionally annotated. Phylogenetic analyses indicated a close relation
to the Red fox (Vulpes vulpes) with an estimated divergence time of ~3.7 million
years ago (MYA). We performed separate enrichment analyses of species-unique genes,
the expanded and contracted gene families, and positively selected genes. The
results suggest an enrichment of pathways related to protein synthesis and response
and an evolutionary mechanism by which cells respond to protein denaturation in
response to heat stress. The enrichment of pathways related to lipid and glucose
metabolism, potentially preventing stress from dehydration, and positive selection
of genes related to vision, as well as stress responses in harsh environments, may
reveal adaptive evolutionary mechanisms in the Corsac fox under harsh drought
conditions. Additional detection of positive selection for genes associated with
gustatory receptors may reveal a unique desert diet strategy for the species. This
high-quality genome provides a valuable resource for studying mammalian drought
adaptation and evolution in the genus Vulpes. School of Life Science, Qufu Normal
University, Qufu 273165, China zhangzhihao1102@126.com; qfxiatian1993@163.com;
zhoushengyang94@163.com; yangxf9066@163.com; 17865717265@163.com;
wanglidong955@163.com; jhfang@qfnu.edu.cn; wangqi907291797@163.com;
douhuashan@163.com; zhanghonghai67@126.com 10.3390/ijms24119599 2023 24
11 - - 9599 -
Hatfield, Robert; Ryder, David; Tidy, Annabel; Hartnell, David; Dean, Karl;
Batista, Frederico Combining Nanopore Sequencing with Recombinase Polymerase
Amplification Enables Identification of Dinoflagellates from the Alexandrium Genus,
Providing a Rapid, Field Deployable Tool Toxins EN Article Alexandrium;
nanopore sequencing; RPA; harmful algal bloom; HABs; saxitoxin; paralytic shellfish
poisoning; in-field sequencing; food safety; aquaculture; VolTRAX The armoured
dinoflagellate Alexandrium can be found throughout many of the world’s
temperate and tropical marine environments. The genus has been studied extensively
since approximately half of its members produce a family of potent neurotoxins,
collectively called saxitoxin. These compounds represent a significant threat to
animal and environmental health. Moreover, the consumption of bivalve molluscs
contaminated with saxitoxin poses a threat to human health. The identification of
Alexandrium cells collected from sea water samples using light microscopy can
provide early warnings of a toxic event, giving harvesters and competent
authorities time to implement measures that safeguard consumers. However, this
method cannot reliably resolve Alexandrium to a species level and, therefore, is
unable to differentiate between toxic and non-toxic variants. The assay outlined in
this study uses a quick recombinase polymerase amplification and nanopore
sequencing method to first target and amplify a 500 bp fragment of the ribosomal
RNA large subunit and then sequence the amplicon so that individual species from
the Alexandrium genus can be resolved. The analytical sensitivity and specificity
of the assay was assessed using seawater samples spiked with different Alexandrium
species. When using a 0.22 µm membrane to capture and resuspend cells, the
assay was consistently able to identify a single cell of A. minutum in 50 mL of
seawater. Phylogenetic analysis showed the assay could identify the A. catenella,
A. minutum, A. tamutum, A. tamarense, A. pacificum, and A. ostenfeldii species from
environmental samples, with just the alignment of the reads being sufficient to
provide accurate, real-time species identification. By using sequencing data to
qualify when the toxic A. catenella species was present, it was possible to improve
the correlation between cell counts and shellfish toxicity from r = 0.386 to r =
0.769 (p ≤ 0.05). Furthermore, a McNemar’s paired test performed on
qualitative data highlighted no statistical differences between samples confirmed
positive or negative for toxic species of Alexandrium by both phylogenetic analysis
and real time alignment with the presence or absence of toxins in shellfish. The
assay was designed to be deployed in the field for the purposes of in situ testing,
which required the development of custom tools and state-of-the-art automation. The
assay is rapid and resilient to matrix inhibition, making it suitable as a
potential alternative detection method or a complementary one, especially when
applying regulatory controls. Centre for Environment Fisheries and Aquaculture
Science, Weymouth DT48UB, UK robert.hatfield@cefas.gov.uk;
david.ryder@cefas.gov.uk; anna.tidy@cefas.gov.uk; david.hartnell@cefas.gov.uk;
karl.dean@cefas.gov.uk; frederico.batista@cefas.gov.uk 10.3390/toxins15060372
2023 15 6 - - 372 -
Fotouhi, Omid; Nizamuddin, Sheikh; Falk, Stephanie; Schilling, Oliver; Knüchel-
Clarke, Ruth; Biniossek, Martin; Timmers, H. Alternative mRNA Splicing Controls
the Functions of the Histone H3K27 Demethylase UTX/KDM6A Cancers EN
Article alternative splicing; histone demethylase; histone methylation;
cancer biology; chromatin; proteomics; bladder cancer The UTX/KDM6A histone H3K27
demethylase plays an important role in development and is frequently mutated in
cancers such as urothelial cancer. Despite many studies on UTX proteins, variations
in mRNA splicing have been overlooked. Using Nanopore sequencing, we present a
comprehensive analysis of UTX/KDM6A splicing events in human cell lines and in
tissue samples from bladder cancer cases and normal epithelia. We found that the
central region of UTX mRNAs encoded by exons 12 to 17 undergoes extensive
alternative splicing. Up to half of all stable mRNAs (8–48% in bladder
tissues and 18–58% in cell lines) are represented by the UTX canonical
isoform lacking exon 14 encoding a nuclear localization sequence, and hence exon
14-containing UTX isoforms exclusively localize to the nucleus, unlike the
cytonuclear localization of the canonical isoform. Chromatin association was also
higher for exon-14-containing isoforms compared to the canonical UTX. Using
quantitative mass spectrometry, we found that all UTX isoforms integrated into the
MLL3 and MLL4, PR-DUB and MiDAC complexes. Interestingly, one of the novel UTX
isoforms, which lacks exons 14 and 16, fails to interact with PR-DUB and MiDAC
complex members. In conclusion, UTX mRNAs undergo extensive alternative splicing,
which controls the subcellular localization of UTX and its interactions with other
chromatin regulatory complexes. Department of Urology, Medical Center-
University of Freiburg, 79106 Freiburg, Germany omid.fotouhi@ki.se; n.sheikh@dkfz-
heidelberg.de; falks@ie-freiburg.mpg.de; oliver.schilling@mol-med.uni-freiburg.de;
rknuechel-clarke@ukaachen.de; martin.biniossek@mol-med.uni-freiburg.de;
m.timmers@dkfz-heidelberg.de 10.3390/cancers15123117 2023 15 12 - -
3117 -
Lee, Hyo-Jeong; Kim, Sang-Min; Jeong, Rae-Dong Analysis of Wheat Virome in Korea
Using Illumina and Oxford Nanopore Sequencing Platforms Plants EN
Article plant virus; wheat; virome; high-throughput sequencing; nanopore
sequencing Wheat (Triticum aestivum L.) is one of the most important staple crops
in the world, along with maize and rice. More than 50 plant viruses are known to
infect wheat worldwide. To date, there are no studies on the identification of
viruses infecting wheat in Korea. Therefore, we investigated virome in wheat from
three different geographical regions where wheat is mainly cultivated in Korea
using Oxford Nanopore Technology (ONT) sequencing and Illumina sequencing. Five
viral species, including those known to infect wheat, were identified using high-
throughput sequencing strategies. Of these, barley virus G (BVG) and Hordeum
vulgare endornavirus (HvEV) were consistently present in all libraries. Sugarcane
yellow leaf virus (SCYLV) and wheat leaf yellowing-associated virus (WLYaV) were
first identified in Korean wheat samples. The viruses identified by ONT and
Illumina sequencing were compared using a heatmap. Though the ONT sequencing
approach is less sensitive, the analysis results were similar to those of Illumina
sequencing in our study. Both platforms served as reliable and powerful tools for
detecting and identifying wheat viruses, achieving a balance between practicality
and performance. The findings of this study will provide deeper insights into the
wheat virosphere and further help improve disease management strategies.
Department of Applied Biology, Institute of Environmentally Friendly
Agriculture, Chonnam National University, Gwangju 61185, Republic of Korea
hjhjhj8@naver.com; kimsangmin@korea.kr; jraed2@jnu.ac.kr
10.3390/plants12122374 2023 12 12 - - 2374 -
Sittikul, Pichamon; Batty, Elizabeth; Yodsawat, Prasert; Nuanpirom, Jiratchaya;
Kosoltanapiwat, Nathamon; Sangket, Unitsa; Chatchen, Supawat; Day, Nicholas;
Thaipadungpanit, Janjira Diversity of Human Enterovirus Co-Circulations in
Five Kindergartens in Bangkok between July 2019 and January 2020 Viruses EN
Article hand–foot–mouth diseases; pediatrics; infectious disease;
Enterovirus A71; whole genome sequencing; SISPA; Oxford Nanopore Technology Human
enterovirus causes various clinical manifestations in the form of rashes, febrile
illness, flu-like illness, uveitis, hand–foot–mouth disease (HFMD),
herpangina, meningitis, and encephalitis. Enterovirus A71 and coxsackievirus are
significant causes of epidemic HFMD worldwide, especially in children aged from
birth to five years old. The enterovirus genotype variants causing HFMD epidemics
have been reported increasingly worldwide in the last decade. We aim to use simple
and robust molecular tools to investigate human enteroviruses circulating among
kindergarten students at genotype and subgenotype levels. With the partial
5′-UTR sequencing analysis as a low-resolution preliminary grouping tool, ten
enterovirus A71 (EV-A71) and coxsackievirus clusters were identified among 18
symptomatic cases and 14 asymptomatic cases in five kindergartens in Bangkok,
Thailand, between July 2019 and January 2020. Two occurrences of a single clone
causing an infection cluster were identified (EV-A71 C1-like subgenotype and
coxsackievirus A6). Random amplification-based sequencing using MinION (Oxford
Nanopore Technology) helped identify viral transmission between two closely related
clones. Diverse genotypes co-circulating among children in kindergartens are
reservoirs for new genotype variants emerging, which might be more virulent or
better at immune escape. Surveillance of highly contagious enterovirus in
communities is essential for disease notifications and controls. Department of
Tropical Pediatrics, Faculty of Tropical Medicine, Mahidol University, Bangkok
10400, Thailand pichamon.sit@mahidol.ac.th; elizabeth.b@tropmedres.ac;
prasert.y@outlook.com; jirath.nuan@gmail.com; nathamon.kos@mahidol.ac.th;
unitsa.s@psu.ac.th; supawat.cht@mahidol.ac.th; nickd@tropmedres.ac;
janjira.tha@mahidol.ac.th 10.3390/v15061397 2023 15 6 - - 1397
-
Fomsgaard, Anna; Tahas, Stamatios; Spiess, Katja; Polacek, Charlotta; Fonager,
Jannik; Belsham, Graham Unbiased Virus Detection in a Danish Zoo Using a Portable
Metagenomic Sequencing System Viruses EN Article point-of-care test
(POCT); human–animal interface; field detection; cross-species transmission;
nanopore sequencing; metagenomic sequencing Metagenomic next-generation
sequencing (mNGS) is receiving increased attention for the detection of new viruses
and infections occurring at the human–animal interface. The ability to
actively transport and relocate this technology enables in situ virus
identification, which could reduce response time and enhance disease management. In
a previous study, we developed a straightforward mNGS procedure that greatly
enhances the detection of RNA and DNA viruses in human clinical samples. In this
study, we improved the mNGS protocol with transportable battery-driven equipment
for the portable, non-targeted detection of RNA and DNA viruses in animals from a
large zoological facility, to simulate a field setting for point-of-incidence virus
detection. From the resulting metagenomic data, we detected 13 vertebrate viruses
from four major virus groups: (+)ssRNA, (+)ssRNA-RT, dsDNA and (+)ssDNA, including
avian leukosis virus in domestic chickens (Gallus gallus), enzootic nasal tumour
virus in goats (Capra hircus) and several small, circular, Rep-encoding, ssDNA
(CRESS DNA) viruses in several mammal species. More significantly, we demonstrate
that the mNGS method is able to detect potentially lethal animal viruses, such as
elephant endotheliotropic herpesvirus in Asian elephants (Elephas maximus) and the
newly described human-associated gemykibivirus 2, a human-to-animal cross-species
virus, in a Linnaeus two-toed sloth (Choloepus didactylus) and its enclosure, for
the first time. Department of Virus & Microbiological Special Diagnostics,
Statens Serum Institut, 5 Artillerivej, 2300 Copenhagen, Denmark anfd@ssi.dk;
st@zoo.dk; ktsp@ssi.dk; chcp@ssi.dk; fon@ssi.dk; grbe@sund.ku.dk 10.3390/v15061399
2023 15 6 - - 1399 -
Maduranga, Sachith; Valencia, Braulio; Sigera, Chathurani; Adikari, Thiruni;
Weeratunga, Praveen; Fernando, Deepika; Rajapakse, Senaka; Lloyd, Andrew; Bull,
Rowena; Rodrigo, Chaturaka Genomic Surveillance of Recent Dengue Outbreaks in
Colombo, Sri Lanka Viruses EN Article dengue; Sri Lanka;
phylogenetics; phylogeography; serotype; genotype; epidemiology All four serotypes
of the dengue virus (DENV1–4) cause a phenotypically similar illness, but
serial infections from different serotypes increase the risk of severe disease.
Thus, genomic surveillance of circulating viruses is important to detect serotype
switches that precede community outbreaks of disproportionate magnitude. A
phylogenetic analysis was conducted on near full length DENV genomes sequenced from
serum collected from a prospective cohort study from the Colombo district, Sri
Lanka during a 28-month period using Oxford nanopore technology, and the consensus
sequences were analyzed using maximum likelihood and Bayesian evolutionary
analysis. From 523 patients, 328 DENV sequences were successfully generated (DENV1:
43, DENV2: 219, DENV3:66). Most circulating sequences originated from a common
ancestor that was estimated to have existed from around 2010 for DENV2 and around
2015/2016 for DENV1 and DENV3. Four distinct outbreaks coinciding with monsoon rain
seasons were identified during the observation period mostly driven by DENV2
cosmopolitan genotype, except for a large outbreak in 2019 contributed by DENV3
genotype I. This serotype switch did not result in a more clinically severe
illness. Phylogeographic analyses showed that all outbreaks started within Colombo
city and then spread to the rest of the district. In 2019, DENV3 genotype I,
previously, rarely reported in Sri Lanka, is likely to have contributed to a
disease outbreak. However, this did not result in more severe disease in those
infected, probably due to pre-existing DENV3 immunity in the community. Targeted
vector control within Colombo city before anticipated seasonal outbreaks may help
to limit the geographic spread of outbreaks. School of Biomedical Sciences, UNSW
Sydney, Sydney, NSW 2052, Australia sarachchige@kirby.unsw.edu.au;
barroyo@kirby.unsw.edu.au; chathuranisigera@med.cmb.ac.lk;
tadikari@kirby.unsw.edu.au; praveen@clinmed.cmb.ac.lk; deepika@parasit.cmb.ac.lk;
senaka@med.cmb.ac.lk; a.lloyd@unsw.edu.au; r.bull@unsw.edu.au;
c.rodrigo@unsw.edu.au 10.3390/v15071408 2023 15 7 - - 1408 -