Microbiological Research 260 (2022) 127023

Contents lists available at ScienceDirect

Microbiological Research
journal homepage: www.elsevier.com/locate/micres

Recovering metagenome-assembled genomes from shotgun metagenomic

sequencing data: Methods, applications, challenges, and opportunities
Yunyan Zhou a, b, *, Min Liu a, Jiawen Yang a
a
State Key Laboratory of Pig Genetic Improvement and Production Technology, Jiangxi Agricultural University, Nanchang 330045, China
b
Institute of Engineering Biology and Health, Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, College of Pharmaceutical Sciences,
Zhejiang University of Technology, Hangzhou, Zhejiang 310014, China

A R T I C L E I N F O A B S T R A C T

Keywords: Reference genomes are essential for analyzing the metabolic and functional potentials of microbiomes. However,
Metagenome-assembled genomes microbial genome resources are limited because most of microorganisms are difficult to culture. Genome binning
Methods is a culture-independent approach that can recover a vast number of microbial genomes from short-read high
Applications
throughput shotgun metagenomic sequencing data. In this review, we summarize methods commonly used for
Challenges
Opportunities
reconstructing metagenome-assembled genomes (MAGs) to provide a reference for researchers to choose pro­
priate software programs among the numerous and complicated tools and pipelines that are available for these
analyses. In addition, we discuss application prospects, challenges, and opportunities for recovering MAGs from
metagenomic sequencing data.

1. Introduction genomic binning approaches from metagenomic data. Genome-binning

Microbial communities play critical roles in many ecosystems. For
example, gut microbial communities are well known to be involved in
host metabolism, immunity, and even behaviors. Reference genomes are
essential for analyzing the potential metabolic and functional capacities
of microbial populations. However, most microorganisms within natural
microbial communities lack available genomes, because many taxa have
not been cultivated in laboratories (Hugenholtz, 2002; Fodor et al.,
2012), despite that significant efforts have been made to culture and
sequence microbiome genomes (Human Microbiome Jumpstart Refer­
ence Strains et al., 2010; Browne et al., 2016; Lagier et al., 2016;
Seshadri et al., 2018; Ito et al., 2019; Zou et al., 2019). Nevertheless, this
lack of information seriously prevents progress into microbiome
research. Single-cell genome sequencing allows researchers to obtain
microbial genomes without cultivation (Marcy et al., 2007). However,
this approach requires special equipment and technical challenges,
including difficulties in isolating single cell and bias in single cell
genome amplification (Gawad et al., 2016).
Numerous advances in high-throughput sequencing technologies
and bioinformatic tools have been made in recent years leading to the
ability of researchers to recover numerous microbial genomes using
groups sequences from the same species or even the same strain together
according to their nucleotide compositions and read coverage from
sequencing data of one or multiple samples. These techniques can pro­
vide insight into the community structures of microbiome at the strain
level (Tyson et al., 2004; Sharon et al., 2013; Sangwan et al., 2016).
Following the first reconstruction of metagenome-assembled genomes
(MAGs) from a natural acidophilic biofilm by Tyson et al. (2004), many
MAGs have been recovered from diverse samples, including from guts
(Wang et al., 2019; Glendinning et al., 2020; Lesker et al., 2020; Chen
et al., 2021a; Feng et al., 2021; Levin et al., 2021; Peng et al., 2021),
rumens (Hess et al., 2011; Svartstrom et al., 2017; Stewart et al., 2019b),
sediments (Anantharaman et al., 2016; Zaremba-Niedzwiedzka et al.,
2017), soils (Hultman et al., 2015; Woodcroft et al., 2018), oceans (Tully
et al., 2018), and other types of environments (Albertsen et al., 2013;
Gullert et al., 2016; Danko et al., 2021). An explosive increase of MAG
numbers has been recovered from human gut communities in recent
years (Almeida et al., 2019, 2020; Nayfach et al., 2019). Indeed, the
Genomes OnLine Database (GOLD) has collected 180,550 isolate ge­
nomes and 18,382 MAGs as of June 2021 (Mukherjee et al., 2019).
Likewise, 258,406 bacterial and archaeal genomes have been taxo­
nomically classified within the Genome Taxonomy Database (GTDB,

* Corresponding author at: State Key Laboratory of Pig Genetic Improvement and Production Technology, Jiangxi Agricultural University, Nanchang 330045,
China.
E-mail addresses: 664280148@qq.com (Y. Zhou), 854768208@qq.com (M. Liu), 383049451@qq.com (J. Yang).

https://doi.org/10.1016/j.micres.2022.127023
Received 18 November 2021; Received in revised form 7 March 2022; Accepted 5 April 2022
Available online 8 April 2022
0944-5013/© 2022 Elsevier GmbH. All rights reserved.
Y. Zhou et al.
Release 06-RS202) as of April 2021, including 45,555 bacterial and
2339 archaeal species clusters (Parks et al., 2020). Nevertheless, the
number of genomes generated thus far considerably surpasses these
numbers. Several studies resulting in large-scale recovery of MAGs have
been conducted in recent years. For example, Anantharaman et al.
(2016) reconstructed 2540 MAGs from an aquifer system, leading to the
generation of genomes from 47 previously uncharacterized
phylum-level lineages. Likewise, Parks et al. (2017) recovered near 8000
MAGs from over 1500 public metagenomic datasets, with a particular
emphasis on environmental and non-human gastrointestinal samples.
Moreover, Stewart et al. (2019b) assembled 4941 rumen microbial
MAGs from 283 ruminant cattle, providing a valuable genomic resource
for evaluating the structures and functions of rumen microbiota. We
previously assembled 6339 MAGs from 500 metagenomes of pig gut
microbiomes, and provided an expanded resource for the studies in
swine gut microbiome (Chen et al., 2021a). Recently, Xie et al. (2021)
assembled 10,373 MAGs from 370 lumen samples comprising ten
gastrointestinal tract regions from seven ruminant species, leading to the
identification of 8745 uncultured candidate species. In humans, Pasolli
et al. (2019) reconstructed 154,723 microbial genomes from human
metagenomes spanning samples from different body sites, host ages,
countries, and lifestyles. In addition, Almeida et al. (2019) generated 92,
143 MAGs from 11,850 human gut metagenomes and identified 1952
uncultured species. They further provided a new catalog comprising
204,938 non-redundant genomes from 4644 gut prokaryotes and con­
structed a Unified Human Gastrointestinal Protein (UHGP) catalog
(Almeida et al., 2020). Researchers have used these assembled
human-associated metagenomes to explore the intraspecies genomic
diversity, leading to the identification of considerable intraspecies
genomic variation that can be specific to populations within human
individuals. Nayfach et al. (2020) reconstructed a genomic catalog of
Earth’s microbiomes from 10,450 metagenomes of diverse habitats, and
that included 52,515 MAGs representing 12,556 novel candidate species
spanning 135 phyla. These recovered MAGs have greatly expanded the
known phylogenetic diversity of bacterial and archaeal communities,
and provided essential resources for the establishment of a taxonomic
framework (Parks et al., 2020). Further, these genomic datasets can
promote thorough investigations into microbial ecological characteris­
tics (Edwards and Holt, 2013; Roller et al., 2013). Moreover, large-scale
MAG data recovery also promote the development of bioinformatic tools
to analyze metagenomic sequencing data, as implemented in the
GTDB-tk (Chaumeil et al., 2019) and PhyloPhlAn3.0 (Asnicar et al.,
2020) software suites.
Many computational algorithms and software programs have been
developed for metagenomic sequencing data analysis (Table S1). How­
ever, a remaining challenge is the suitable choice of software or pipeline
programs among numerous bioinformatic tools, and especially for re­
searchers without bioinformatics backgrounds. Here, we summarize
available methods and software programs that are commonly used to
construct MAGs, while also discussing the advantages and limitations of
these programs for MAG generation from short-read shotgun meta­
genomic sequencing data. We further discuss application, challenges
and opportunities for MAG generation in microbial studies.
2. Procedures and software for reconstructing microbial
genomes from metagenomic sequencing data
A typical shotgun metagenomics study contains five steps including
study design, sample collection and sequencing, pre-processing of
sequencing data, sequence analysis, and post-processing and validation
(Quince et al., 2017). Recovering MAGs is an effective approach in
metagenomics studies for mining useful information from metagenomic
sequencing data. Saheb Kashaf et al. (2021) provided a protocol to
recover draft genomes from short read shotgun metagenomic
sequencing data. A conventional workflow for reconstructing microbial
genomes from metagenomic sequencing data primarily includes the
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Microbiological Research 260 (2022) 127023
following three modules (Fig. 1): (i) preprocessing of sequence reads
including adapter trimming, quality control, and host genomic sequence
removal; (ii) reconstruction of MAGs including metagenomic assembly,
metagenomic binning, and optimization of MAG quality; (iii) quantifi­
cation and annotation of MAGs including quantification, taxonomic
classification, and genome annotation. Based on study purposes, MAGs
can then be used for one of further analyses, such as functional capacity
analysis, phylogenetic analysis, biomarker discovery, pan-genome
analysis, identification of single nucleotide polymorphisms (SNPs) at
the population level, and host prediction of viral genomes. To evaluate
the selection of appropriate software programs among various and
complicated tools and pipelines, an understanding of the algorithms is
first needed along with a comparison of the advantages and limitations
of these programs.
2.1. Sequence reads preprocessing
High-throughput sequencing platforms allow multiple samples to be
sequenced at once by adding dual indexed barcodes during library
preparation to distinguish sequence reads from each sample. Sequence
errors, base content biases, and overrepresented sequences are all pro­
duced during library preparation and sequencing. Indeed, from sample
collection to DNA extraction, library preparation, and sequencing,
contaminants can be introduced at each step from hosts, humans or
surrounding environments. Therefore, sequence read preprocessing is
important for further analyses. Several software programs are available
for adapter trimming and quality control (Table 1). Trimmomatic is a
popular tool that exhibits good performance in trimming poor-quality
reads, but it is only operational for Illumina platforms sequence data
(Bolger et al., 2014). In addition, Trim Galore (https://www.bioinforma
tics.babraham.ac.uk/projects/trim_galore/) incorporates FASTQC
(Andrews, 2010) and Cutadapt (Martin, 2011), and can be used with
data from all high-throughput sequencing platforms for trimming low
quality and adapter sequences. Further, Fastp can automatically identify
adapters, and rapidly perform quality control and adapter trimming,
producing results in both the JSON and HTML formats (Chen et al.,
2018). Removal of contaminant reads can be performed by aligning
reads to reference genome sequences including host genomic sequences.
BWA (Li and Durbin, 2009) and Bowtie 2 (Langmead and Salzberg,
2012) are two of the mostly commonly used tools for alignment of short
sequence reads to reference genomes. In addition, the software pro­
grams for file format transformation such as Samtools (Li et al., 2009)
and Bedtools (Quinlan and Hall, 2010) are needed sometimes. SeqKit is a
toolkit for FASTA/Q file manipulation that supports the production of
simple statistics, file format conversions, searching, filtering, extraction,
deduplication, splitting, shuffling, and sampling (Shen et al., 2016).
Further, the MultiQC program has traditionally been used to summarize
and report results from multiple tools and samples within a single file
(Ewels et al., 2016). KneadData (https://github.com/biobaker­
y/kneaddata) is a new pipeline that incorporates all of the above func­
tions including in quality control, adapter trimming, and removing host
genomic sequences, thereby making it a useful pipeline for preprocess­
ing metagenomic and metatranscriptomic sequencing data.
2.2. Metagenome assembly
Metagenome assembly involves de novo assembly of short meta­
genomic sequence reads into contigs. Popular graph-based algorithms
for metagenome assembly include Overlap-Layout-Consensus (OLC) and
de Bruijn graph (DBG) algorithms (Miller et al., 2010; Perez-Cobas et al.,
2020). DBG algorithms based on k-mer compositions are the most
commonly used approaches in short read metagenomic assemblers
(Pevzner et al., 2001). In addition, OLC algorithms can be used to
reconstruct longer contigs based on overlap between paired reads.
However, this approach has a high error rate and has rarely been used
with short read data. Nevertheless, the emergence of long-read
Y. Zhou et al. Microbiological Research 260 (2022) 127023

Fig. 1. | Typical workflow for reconstructing and analyzing metagenome-assembled genomes (MAGs) from metagenomic sequencing data. The steps for recon­
structing and analyzing MAGs includes: (i) preprocessing sequencing reads including adapter trimming, quality control, and host genomic sequence removal; (ii)
MAG reconstructions including metagenome assembly, metagenomic binning, and optimization of MAG qualities; (iii) quantification and annotation of MAGs
including quantification, taxonomic classification, and genome annotation; and (iv) advanced MAG analyses.

for sequence assembly. Several studies have evaluated the performance

Table 1
of multiple de novo assemblers using metagenomic sequencing data
Software programs used for preprocessing raw sequence reads.
from distinct environments and simulated datasets (Sczyrba et al., 2017;
Software Major functions Description and advantages van der Walt et al., 2017; Forouzan et al., 2018). Among the numerous
FastQC Quality assessment Identifies potential problems available assemblers, metaSPAdes and MEGAHIT are most recom­
Summary report production mended. metaSPAdes was updated from SPAdes (Bankevich et al.,
Cutadapt Quality control, adapter Removes unwanted sequence 2012), with the former being specific for metagenomic sequencing data,
trimming
Trimmomatic Quality control, adapter Good performance in trimming poor-
and exhibits excellent performance in assembling long contig lengths,
trimming quality data with a high fraction of reads assembled into contigs. However, meta­
Contains a library of Illumina SPAdes also incurs increasing mis-assemblies, and is both time and
adapters and primer sequences memory intensive. MEGAHIT is an alternative choice if both a high level
Used for Illumina platform sequence
of assembly completeness and low number of mis-assemblies are
data
Trim galore Quality control, adapter A wrapper tool for FastQC and desired. MEGAHIT features advantages of fast assembly and low mem­
trimming Cutadapt ory requirements. Further, a high level of accuracy can be achieved by
Automatic identification of adapters MEGAHIT for assemblies from metagenomic sequencing data with low
fastp Quality control, adapter Fast complexity. The Critical Assessment of Metagenome Interpretation
trimming Automatic identification of adapters
Summary report production
(CAMI) (Sczyrba et al., 2017) study indicated that MEGAHIT, Minia and
KneadData Quality control, adapter A pipeline that integrates multiple Meraga (Meraculous (Chapman et al., 2011) + MEGAHIT) obtained
trimming, removing host tools, including Trimmomatic, higher cumulative contig sizes and contig numbers than the OperaMS
genomic sequence FastQC and Bowtie2 Scaffolder (Gao et al., 2011), Ray Meta (Boisvert et al., 2010) and the
Designed for metagenomic and
Velour assembler (Zerbino and Birney, 2008). The IDBA-UD assembler
metatranscriptomic sequencing data
features good performance with highly uneven sequence data depth
(Peng et al., 2012).
sequencing technology has led to re-popularization of OLC algorithms. AMOS (Treangen et al., 2011) and MeGAMerge (Scholz et al., 2014)
Many assemblers are available based on DBG algorithms (Table 2), are shotgun metagenome assemblers that use the OLC approach, and
including the metaSPAdes (Nurk et al., 2017), MEGAHIT (Li et al., combine the results of multiple assemblers to improve assembly quality.
2015), SOAPdenovo2 (Luo et al., 2012), IDBA-UD (Peng et al., 2012), Further, the GAM-NGS (Vicedomini et al., 2013) program is also capable
and Minia (Chikhi and Rizk, 2013) assemblers. All of these software of merging multiple assemblies to improve contiguity and correctness.
programs exhibit multi-k-mer capabilities that are very typically used However, the program does not rely on global alignment to build

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Y. Zhou et al. Microbiological Research 260 (2022) 127023

Table 2 QUAST (Gurevich et al., 2013) can detect mis-assemblies and structural
Popular software programs for metagenomic assembly. variations using a reference-based approach, while also evaluating
Program Assembly Algorithms Descriptions and Advantages contig sizes and genome representation, even without a reference.
MetaQUAST (Alla et al., 2016) is a modified version of QUAST that is
MEGAHIT De Bruijn graphs Less computational resources
Multi-k assembly Fewer mis-assemblies specifically designed for metagenomic sequence assembly, and that
Bias towards low coverage features additional functionalities. Specifically, the former can detect
genomes chimeric contigs and unknown species contents. In addition, the Deep­
metaSPAdes De Bruijn graphs Long contig lengths MAsED (Mineeva et al., 2020) program uses a deep learning approach to
Multi-k assembly High fraction of reads that can
be assembled to contigs
identify mis-assembled contigs independently from reference genomes.
SOAPdenovo2 De Bruijn graphs Less computational resources Different programs carry different time and memory consumption needs
Multi-k assembly needed and these should be considered when choosing programs for quality
Greatly surpasses its predecessor assessment.
SOAPdenovo in both assembly
Metagenomic sequence assembly strategies include single-sample
length and accuracy
IDBA-UD De Bruijn graphs Better performance for assembly and co-assembly of multiple samples (Table 3). Co-assembly
Multi-k assembly sequencing data with highly can lead to obtaining more genomes with higher completeness, and
uneven depths more genomes from the species exhibiting low abundances, but also
Minia De Bruijn graphs Less computational resources leads to higher contamination (Pasolli et al., 2019). To increase the
Multi-k assembly needed
Canu Overlap-Layout-Consensus Long-read assembler
fraction of reads that can be assembled to contigs and the accuracy of
AMOS Overlap-Layout-Consensus Merges results from multiple assemblies, a combined approach of single-sample assembly and
assemblers to improve overall co-assembly of multiple samples should be considered (Stewart et al.,
assembly quality 2019b). However, increased data sizes and their requirements of large
MeGAMerge Overlap-Layout-Consensus Merges assembled contigs, long
computational resources lead to the co-assembly approach not being
reads from metagenomic
sequencing runs suitable for analyzing large datasets with large sample sizes. Genomes
GAM-NGS Identifies regions of two Less computational resources produced from co-assembly should be considered as population-level
assemblies representing the needed genomes. Consequently, assembled contig quality may decrease when
same genomic locus by read Merges results from multiple sequence reads from different sample types are included (Saheb Kashaf
alignment and stores them in a assemblers to improve overall
et al., 2021). A previous study also suggested that single-sample as­
weighted graph assembly quality
MaSuRCA Overlap-Layout-Consensus Supports assemblies of short sembly can lead to a greater number of higher quality genomes
and de Bruijn graphs reads together with long reads compared to co-assembly (Olm et al., 2017).
Suitable for low-complexity De-replication of MAGs must be conducted when pooling samples,
databases
with dRep being a recommended de-replication tool that performs
hybridSPAdes De Bruijn graphs Supports assemblies of short
reads together with long reads pairwise genome comparisons based on average nucleotide identity
OPERA-MS De Bruijn graphs and Supports assemblies of short (ANI). The highest quality genome is then chosen from each replication
scaffolding reads together with long reads cluster to generate a de-replicated catalog of MAGs (Olm et al., 2017).
Long-read connections Strain-resolved assembly However, increased MAG numbers lead to dramatic increases in
required computational resources and time for de-replication. Mash is
assembly graphs, but rather identifies regions of two assemblies that another tool for de-replicating MAGs that exhibits fast computational
represent the same genomic locus by read alignment, followed by stor­ speeds and estimates the similarity between genomes using the MinHash
age in a weighted graph. Canu (Koren et al., 2017) is an assembler that is distance (Ondov et al., 2016). However, high MAG completeness is
specially designed for assembling long reads generated from long-read required for MAGs this analysis.
sequencing technologies like those from PacBio or Oxford Nanopore
methods. Hybrid assembly methods combine the advantages of 2.3. Metagenome binning
high-accuracy second-generation sequencing data with the long
sequence lengths of third-generation sequencing data. The hybrid Metagenomic binning groups sequences from the same species or
approach ultimately provides higher quality contigs than using either even the same strain into genome bins. Binning can be conducted via
short read or long read assemblies alone, thereby uniquely in resolving read, contig, or gene binning based on the type of sequence data that are
complex repeated DNA segments. Several hybrid assemblers have been clustered (Nielsen et al., 2014; Girotto et al., 2016; Sangwan et al.,
developed in recent years including MaSuRCA (Zimin et al., 2017), 2016). Binning contigs have been most commonly used for genome
hybridSPAdes (Bankevich et al., 2012) and OPERA-MS (Bertrand et al., binning in recent years. Generally, MAGs are generated by contig
2019). binning into taxonomic bins based on nucleotide compositions or
Metagenomic sequence assembly is challenged by many factors sequence read abundances. The tools commonly used for binning pri­
including intragenomic and intergenomic repeats, uneven species marily include MetaBAT 2 (Kang et al., 2019), Maxbin 2 (Wu et al.,
abundances, uneven sequence coverage, high strain diversity, low re­ 2016) and CONCOCT (Alneberg et al., 2014) (Table 4). MetaBAT 2 has
covery rates for some phyla, and sequencing errors (Nayfach et al., 2019;
Olson et al., 2019). Further, assembly quality can be influenced by Table 3
community complexity, the presence of closely related genomes, Comparison of single sample assembly and co-assembly of multiple samples.
sequencing depth, and k-mer sizes (Sczyrba et al., 2017). Consequently,
Assembly Advantages Disadvantages
the accurate assessment of metagenome assembly qualities is critical for strategy
further analysis. Indicators for assembly quality primarily include the
Single- Lower contamination Lower completeness
following three aspects: (i) statistical indicators including the numbers assembly Fast Difficult to obtain genomes from
of contigs, average lengths of contigs, and N50 values; (ii) the accuracy Memory efficient low-abundance species
of assembled contigs including mismatches and the number of Suitable for analyses with
mis-assemblies; and (iii) completeness of contigs as measured by the large sample sizes
Co-assembly Higher completeness Higher contamination
fraction of reads that are mapped to them (Forouzan et al., 2018).
Obtains more genomes from Time-consuming
Several software programs are available for assessing assembly quality. low-abundance species High memory requirements

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Y. Zhou et al. Microbiological Research 260 (2022) 127023

been commonly recommended for MAG reconstructions from large

Bins are then combined from more than three binning results from programs (e.g., from CONCOCT, MetaBAT, and MyCC).
An adaptive binning algorithm (using abundance (ABD) to rank-normalize tetranucleotide frequencies (TNF) and calculate
sample size datasets because of its highly efficient use of computational

Iteratively compares bin sets and selects a better bin based on the scoring function S=Completion-5 *Contamination for
memory. The numbers and quality of assembled bins are greatly influ­

Combines the bins from multiple binning results (e.g., CONCOCT, MetaBAT, MaxBin2, tetraESOMs, and ABAWACA).
enced by contig length, and thus, most of the aforementioned programs

The Binning_refiner module is used to produce possible combinations of bin sets from multiple binning results.
An iterative procedure is used based on bin score, scaffold N50 and bin size to select a non-redundant bin set.
suggest the use of contigs with ≥ 1000 bp for binning. However, contig

Uses a Gaussian mixture model fitting with a variational Bayesian approximation to cluster contigs into bins.

A scoring function to rank genome bins is used based on their estimated completeness and contamination.
lengths must be ≥ 1500 bp when MetaBAT 2 is used for binning. Vari­
ational Autoencoders for Metagenomic Binning (VAMB) is a recently
a composite score of TNF and ABD; graph-based clustering; small contigs bin recruitment is possible).

developed software program for metagenomic binning (Nissen et al.,

Determines clustering threshold based on the density of Pearson correlation coefficient distances.
Uses variational autoencoder (VAE) to encode abundances and compositions of contigs or genes.
2021) and encodes sequences according to abundances and k-mer dis­
tribution information before clustering bins using deep variational
autoencoders. VAMB usually obtains fewer bins compared with Meta­

Removes contigs with divergent GC content, coverage, or tetranucleotide signatures.

BAT 2, but the number of bins is relatively stable and robust when using
different minimum contig length thresholds.
Uses an Expectation-Maximization algorithm to cluster contigs into bins.

Pairwise BLASTn is used to get shared contigs between two sets of bins. 2.4. Optimization of MAG quality
Initializes the number of genomes based on single copy maker genes.

No uniform criteria for MAG quality have yet been established,

Uses iterative medoid clustering of contigs or genes into bins.

although estimated completeness and contamination are two major in­

A simple k-means algorithm is used to initialize the clusters.

dicators of MAG quality. CheckM (Parks et al., 2015) can be used to

estimate completeness and contamination of bacterial or archaeal ge­
Removes contigs with incongruent 16 S rRNA genes.
nomes using lineage-specific marker genes by referencing an established
genome tree. CheckM has been used to assess MAG quality in almost all
Removes contigs with conflicting taxonomy.

studies that recover MAGs from shotgun metagenomic sequencing data.

However, CheckM is unable to assess the quality of non-bacterial or
Regularized expectation maximization.

archaeal genomes, including those from fungi or other microbial eu­

karyotes (Parks et al., 2015). In contrast, the BUSCO (Manni et al., 2021)
program can be used to assess MAG qualities from bacterial, archaeal,
viral, and eukaryotic species. Moreover, the program is suitable for
various data types including genome assemblies, gene sets and MAGs.
each pair of bins.

The DAS Tool (Sieber et al., 2018), Binning_refiner (Song and

Thomas, 2017), or MetaWRAP (Uritskiy et al., 2018) programs can all be
Algorithm

used to refine bins to achieve higher completeness and less contamina­

tion (e.g., refining towards higher quality) by integrating several
binning results. In addition, RefineM can be used to identify and remove
contigs with divergent GC content, coverage, and tetranucleotide sig­
Groups contigs into individual bins based on tetranucleotide frequencies and

Groups contigs into individual bins based on tetranucleotide frequencies and

Refines bins by integrating the results of multiple binning software programs.

Refines bins by integrating the results of multiple binning software programs.

A pipeline for recovering MAGs and refining bins by integrating the results of

natures, in addition to those with conflicting taxonomy, and incongruent

(tetranucleotide by default) and contig coverages across multiple samples.

16 S rRNA genes (Parks et al., 2017). Genome bins can also be further
Refines bins by removing contigs within a bin that were identified as

refined by extracting reads that belong to each bin and then

re-assembling them using metaSPAdes (Nurk et al., 2017).
Groups contigs into individual bins based on k-mer frequencies

MAG quality has also been evaluated using several other metrics
including strain heterogeneity, quality score (com­
pleteness − 5 × contamination), the number of contigs within a MAG,
the presence of rRNA genes (23 S, 16 S, and 5 S), and the number of
tRNAs (Parks et al., 2017; Pasolli et al., 2019; Stewart et al., 2019b;
Bins microbial genomes using deep learning.

Nayfach et al., 2020). The Minimum Information about a Metagenome

contig coverages across multiple samples.

contig coverages across multiple samples.

Assembled Genome (MIMAG) standards have been proposed by the

multiple binning software programs.

Genomic Standards Consortium, stating that MAGs with more than 50%
Software programs used for binning and refining genome bins.

completeness and less than 10% contamination are considered as

medium-quality. In addition, high-quality MAGs are considered those
with more than 90% completeness, less than 5% contamination, and the
presence of all 23 S, 16 S, and 5 S rRNA genes, in addition to at least 18
tRNAs (Bowers et al., 2017).
contamination.
Description

2.5. Quantification and annotation of MAGs

Medium- or high-quality MAGs are commonly subjected to quanti­

fication, taxonomic classification and genome annotation. MetaWRAP is
Binning_refiner

a recommended pipeline for those analyses if sample sizes are not too
MetaWRAP
MetaBAT 2

CONCOCT

large. As indicated above, MetaWRAP is a flexible and modular pipeline

Maxbin 2

DAS Tool
Software

RefineM
VAMB

that comprises all steps from preprocessing of metagenomic sequence

reads to the recovery of MAGs, in addition to quantification and anno­
tations of MAGs. However, high computational capacity is required to
refinement

run MetaWRAP. Anvi’o (Eren et al., 2015) is another analytics and

visualization platform for ‘omics data, that can also perform assembly,
Binning
Table 4

mapping, profiling, binning, refinement of bins (with interactive in­

Bins

terfaces), and summarize results. In addition, MAGpy (Stewart et al.,

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Y. Zhou et al. Microbiological Research 260 (2022) 127023

2019a) is another pipeline for downstream analyses of MAGs including (Chaumeil et al., 2019), MiGA (Rodriguez et al., 2018), PhyloPhlAn
comparison of protein or genome sequences between MAGs and several 3.0 (Asnicar et al., 2020) and MAGpy (Stewart et al., 2019a) programs.
public databases, quality assessment, taxonomic classification, and Among these, GTDB-tk has been most widely used among many studies
phylogenetic analysis. Further, Salmon (Patro et al., 2017) has long been in recent years (Danko et al., 2021; Nayfach et al., 2020; Xie et al., 2021;
used to quantify the abundances of RNA-seq data transcripts, and has Chen et al., 2021a).
also been used to quantify MAGs abundances. The quantification mod­ Prokka (Seemann, 2014) is a pipeline that integrates multiple soft­
ule of MetaWRAP calculates the abundances of each MAG in each ware programs to annotate MAGs. The program can predict coding
sample based on the length-weighted average of the contig abundances sequence (CDS), rRNA genes, and tRNA gene from MAGs. The output
that are generated by Salmon (Uritskiy et al., 2018). files generated by Prokka include FASTA files of protein and nucleotide
MAG taxonomic classification methods can be classified into three sequences of coding genes, a Genbank file, and a GFF (v3) file containing
categories: (i) DNA-based classification including through average sequences and annotations. All of these files can be easily used in other
nucleotide identity (ANI) and alignment of genome sequences; (ii) downstream analyses.
protein-based classification based on average amino acid identity (AAI)
and alignment of protein sequences; and (iii) marker-based classification
based on species-specific core gene datasets and universal markers 2.6. Public microbial genome databases
(Table 5). The classification module of MetaWRAP (Uritskiy et al., 2018)
uses DNA-based classification, while BAT (von Meijenfeldt et al., 2019) To construct more complete microbial genome datasets, researchers
classifies MAGs using protein-based classification. Some tools also typically integrate MAGs with public microbial genome databases. Mi­
classify MAGs using more than two methods, including the GTDB-tk crobial genome databases that are available and that are commonly used
for taxonomic annotation include the Genome Taxonomy Database
(GTDB) (Parks et al., 2020), Integrated Microbial Genomes (IMG) (Chen
Table 5
Software used to taxonomic classification of MAGs.
et al., 2021b), RefSeq (https://www.ncbi.nlm.nih.gov/refseq/), and
GenBank (https://www.ncbi.nlm.nih.gov/genbank/) databases. In
Software Database Classification methods Type
addition, public reference genome datasets from specific environments
GTDB-tk Genome Taxonomy Placement in the GTDB DNA-based, or from isolate genomes include the Human Microbiome Project (HMP)
Database (GTDB) reference tree marker-
(Human Microbiome Jumpstart Reference Strains et al., 2010), the
Relative evolutionary based
divergence (RED)
Genomic Encyclopedia of Bacteria and Archaea (GEBA) (Mukherjee
Average nucleotide et al., 2017), the Genomes from Earth’s Microbiomes (GEM) (Nayfach
identity (ANI) et al., 2020), rumen-uncultured genomes (RUGs) (Stewart et al., 2019b),
MiGA NCBI Genome Database Average nucleotide DNA-based,
(Prokaryotes), RefSeq identity (ANI) protein-
Average amino acid based Table 6
identity (AAI) Genome databases or public reference genome datasets commonly used for
PhyloPhlAn 87,173 reference Comparison against the DNA-based, genome comparisons.
3.0 genomes in GenBank, 400 most universal marker-
Database/ # Genomes # Species Web Address Last update
154,723 MAGs, markers across based
Dataset time/
57,841,793 gene bacterial and archaeal
version
families in UniRef90 species
Species-specific core GTDB Bacteria: Bacteria: https://gtdb.ecog April 27,
genes from the 254,090 45,555 enomic.org/ 2021/
> 18,000 sets of Archaea: Archaea: Release 06-
preselected gene 4316 2339 RS202
families in UniRef90 IMG Bacteria: Bacteria: https://img.jgi. June 5,
Average nucleotide 93,966 32,334 doe.gov/cgi-bin/w 2021/
identity (ANI) Archaea: Archaea: /main.cgi Version
MAGpy UniProt, over 100,000 Comparison of protein DNA-based, 2114 1060 5.4.1
public genomes sequences protein- RefSeq Bacteria: Bacteria: https://ftp.ncbi. Jan. 9,
Comparison of genome based, 203,019 31,179 nlm.nih.gov/ge 2021
sequences marker- Archaea: Archaea: nomes/refseq/
Uses PhyloPhlAn based 1107 713
(based on the 400 most GenBank Bacteria: Bacteria: https://ftp.ncbi. Jan. 9,
universal markers) to 833,917 51,841 nlm.nih.gov/g 2021
perform phylogenetic Archaea: Archaea: enomes/genbank/
analysis 6274 2418
MetaWRAP NCBI Nucleotide Taxonomic DNA-based HMP 2947 / https://www.ncbi. Published
Sequence Database classification of each nlm.nih.gov/bi in 2010
(NT), NCBI Taxonomy contig oproject/28331
Database, RefSeq The final taxonomy of a GEBA Bacteria:974 579 https://genome. Published
bin is determined based Archaea: 29 genera jgi.doe.gov/porta in 2017
on the taxonomy of l/geba1003/geba
each contig in a 1003.info.html
phylogenetic tree and GEM 52,515 18,028 https://genome.jg Published
the branch weight i.doe.gov/portal/ in 2020
BAT NCBI non-redundant ORF prediction Protein- GEMs/GEMs.
protein database (NR), Predicted ORFs are based home.html
NCBI Taxonomy aligned to the NCBI_nr RUGs Bacteria: 2346 https://www.ebi. Published
Database database 4815 ac.uk/ena/brow in 2019
ORFs are classified Archaea: ser/view
based on the LCA 126 /PRJEB31266
algorithm Hungate1000 410 82 genera https://genome. Published
MAGs are then collection jgi.doe.gov/port in 2018
classified based on a al/HungateCollect
voting approach using ion/HungateColle
all classified ORFs ction.info.html

6
Y. Zhou et al. Microbiological Research 260 (2022) 127023

and the Hungate genome catalog (Seshadri et al., 2018) (Table 6). There Table 7
are, of course, more public microbial genome datasets than those listed Databases commonly used to functional annotate MAGs.
above, individual researchers can choose their databases according to Database Description Item Web Address Analysis tools
specific environments and the purpose of the study. The comprehen­
KEGG High-level KEGG https://www BLAST (
siveness and taxonomic accuracy of a reference genome database can functions and Orthology .kegg.jp/ Altschul et al.,
also critically impact the accuracy of MAG taxonomic annotation. Un­ utilities of (KO), modules 1990),
known taxa can be identified by taxonomic classification and by biological and pathways GhostKOALA
comparing sequence similarities between recovered genomes and public systems (Kanehisa
et al., 2016)
reference genomes. CAZy Carbohydrate Carbohydrate- http://www. dbCAN (Yin
It should be noted that the definition of bacterial species remains metabolism active cazy.org/ et al., 2012)
controversial. A species can be classified based on several aspects, enzymes enzymes
including monophyly, ribosomal RNA genes, genomic coherence and (CAZymes)
eggNOG Orthologous Orthologous http://eggno eggNOG-
phenotypic coherence (Rossello-Mora and Amann, 2015). Thus,
relationships, groups (OGs) g.embl.de mapper (
sequence similarity may be different for each taxonomy, even when gene Huerta-Cepas
considering the same taxonomic level. Consequently, it is difficult to evolutionary et al., 2017)
accurately classify MAGs to the species level using a fixed threshold for histories and
sequence similarity. Generally, the recovered genomes can be clustered functional
annotations
to strain-level bins and species group bins (SGBs) at the thresholds of antiSMASH Secondary BGCs https://antis antiSMASH (
99% and 95% ANI, respectively. Nevertheless, high quality is required to metabolite mash.secon Blin et al.,
obtain high taxonomic accuracy for MAGs. ‘biosynthetic darymetabo 2019)
gene clusters’ lites.org
(BGCs)
3. Advanced analyses of MAGs
CARD Reference DNA Antibiotic http:// BLAST (
and protein resistance arpcard.mcm Altschul et al.,
Genomes of uncultured and previously uncharacterized microbial sequences, genes (ARGs), aster.c 1990), RGI(
taxa can be obtained by metagenomic binning. These recovered ge­ detection Antibiotic Alcock et al.,
nomes can then provide new resources for microbial genome databases. models, and Resistance 2020)
bioinformatics Ontology
Thus, advanced analyses, including through functional analysis, tools for the (ARO)
comparative genomic analysis, and host prediction of viral genomes, can molecular basis
yield valuable biological insights from MAGs. of bacterial
antimicrobial
resistance
3.1. Potential functional capacities of microbiota
(AMR)
VFDB Virulence VFs http://www. BLAST (
MAGs can be used to further explore the potential functional ca­ factors (VFs) of mgc.ac.cn/ Altschul et al.,
pacities of microbiota. For example, Stewart et al. (2019b) constructed a bacterial VFs/ 1990),
gene catalog containing 10.69 million predicted genes from > 5000 pathogens. VFanalyzer (
Liu et al.,
rumen MAGs and isolate genomes, leading to the identification of 442, 2019)
917 genes involved in carbohydrate metabolism. A large percentage of
these predicted CAZymes exhibited low sequence similarity to those in
the CAZy database. This catalog consequently provides an important (Table 7).
gene resource for encoded proteins and enzymes in the study of rumen
microbiomes. Nayfach et al. (2020) also constructed a catalog contain­ 3.2. Evolution and variation of microbiota
ing 111,428,992 full-length genes that were classified into 5794,145
protein clusters (PCs) as part of the Earth’s Microbiomes (GEM) catalog. Extensive strain diversity within species suggests that genomic in­
Among these PCs, nearly 70% were newly identified and represent new formation from a single genome is insufficient to reflect the gene pool
functional capacities. In addition, 87,187 novel biosynthetic gene clus­ and functional capacities for a species. Increased numbers of microbial
ters (BGCs) were identified and subjected to in depth analysis of their genomes allow comparative genomic analysis to become a powerful
biosynthetic potentials. method to understand the relatedness and genomic variation among
Importantly, microbial cultivation is an effective method for exper­ strains within species. Comparative genomics analyses includes evalu­
imental verification of microbial functions, but can also expand the ating phylogenetic relationships among taxa (Pasolli et al., 2019; De
reservoir of known taxa and functions inferred by metagenomic analyses Filippis et al., 2020), genome-wide comparisons (Koonin and Mushe­
(Lagier et al., 2018; Liu et al., 2020). Vice versa, MAGs can provide gian, 1996; Koonin et al., 1996), identification of SNPs (Nayfach et al.,
information for improving cultivation conditions via analysis of ge­ 2019) and pan-genome analysis (Medini et al., 2005; Bezuidt et al.,
nomes, thereby increasing the number of bacterial strains that are suc­ 2016; Pasolli et al., 2019). Karcher et al. (2020) suggested that microbial
cessfully isolated and cultivated (Nayfach et al., 2019). Previous studies population structures are associated with geography and the evolu­
of MAGs have suggested uncultured bacteria can exhibit small genome tionary relationships of their host based on comparative genomic anal­
sizes and slow replication rates, while also lacking many genes associ­ ysis of 1321 Eubacterium rectale genomes recovered from human gut
ated with cultivated organisms due to the loss of numerous relatively metagenomes spanning geographic origins and host lifestyle. Likewise,
conserved metabolic pathways (Brown et al., 2015, 2016; Nayfach et al., De Filippis et al. (2020) used comparative genomics to show that
2019). Databases commonly used for functional annotations of MAGs functional potential and diversity vary among 3000
include the Kyoto Encyclopedia of Genes and Genomes (KEGG) (Kane­ Faecalibacterium-like MAGs based on host age, origin, lifestyle, and
hisa and Goto, 2000), carbohydrate-active enzymes (CAZy) database disease. Tett et al. (2019) explored the global population structure and
(Lombard et al., 2014), evolutionary genealogy of genes non-supervised genetic diversity of Prevotella copri using 1023 genomes (comprising 17
orthologous groups (eggNOGs) (Huerta-Cepas et al., 2019), antibiotics sequenced isolates and 1006 MAGs). In addition to the above, several
and secondary metabolite analysis shell (antiSMASH) (Blin et al., 2021), studies have shown that horizontal gene transfer is a dominant force in
Comprehensive Antibiotic Resistance Database (CARD) (Alcock et al., prokaryotic evolution. Comparative genomics can provide a deeper
2020) and the Virulence Factor Database (VFDB) (Chen et al., 2016) understanding of the source of genes from different microbial species at

7
Y. Zhou et al.
the genomic level (Koonin and Wolf, 2008; Bezuidt et al., 2016).
Moreover, comparative genomics can help to determine the presence or
absence of genes with important functions (e.g., antibiotic resistance
genes and virulence genes) (Edwards and Holt, 2013).
3.3. Host prediction for viral genomes
Viruses play significant roles in global ecosystem functioning (Suttle,
2007; Shkoporov and Hill, 2019). Moreover, viruses are estimated to be
the most abundant microbial entities on Earth and out-number their
prokaryotic hosts by over an order of magnitude. However, the micro­
bial hosts of viruses are largely unknown, and especially for uncultivated
viruses. MAGs can be valuable resources for investigating of virus-host
interactions. Many approaches exist that enable the prediction of pre­
dict bacteriophage-bacteria relationships, including CRISPR-spacer
matching, genetic homology matcheing, and abundance profile com­
parisons (Edwards et al., 2016). Thousands of interactions between vi­
ruses and bacteria have been identified from MAGs using these
approaches (Nayfach et al., 2020; Danko et al., 2021) and provide
considerable knowledge for the understanding for interactions between
phages and their host bacteria.
4. Application prospects of MAGs
MAGs have huge application prospects in human, animal and other
environments via their combination with other data including applica­
tion as clinical indicators, diagnosing disease status, host phenotypes,
and for other omics data (including culturomics and metabolomics). In
contrast to traditional culture-based methods and 16 S rRNA sequencing
methods, the assembly of microbial genomes through metagenomics can
simultaneously detect diverse bacteria, archaea and viruses, and also
reveal the functions of microorganisms at the strain or even genomic
levels. Here, we introduce some potential application areas and specific
application examples of MAGs in human, animal and other
environments.
4.1. Application prospects of MAGs in human studies
Microbiomes play important roles in human health and under­
standing the function of these microbial communities along with their
specific strains can provide biotechnology prospects for therapeutic
fields and open up new potential treatments of diseases. Specifically,
strain-level resolution of MAGs can help to promote infectious disease
diagnosis, microbiome analyses in diseased and healthy states, pre­
dictions of antimicrobial resistance (Bradley et al., 2015; Lo et al.,
2018), detection of virulence determinants (Teh et al., 2021; Wang et al.,
2021), and disease biomarker identification (Sommer et al., 2017; Dapa
et al., 2022). Phages have been used to treat human diseases due to
growing antimicrobial resistance in the post-antibiotic age (Dedrick
et al., 2019; Sabino et al., 2020; Hsu et al., 2021). To this end, MAGs are
important resources for identifying host bacteria-phage associations and
provide valuable information for developing phage therapy (Fujimoto
et al., 2020).
Microbiota exhibit important influences on drug metabolism and
therapeutic effects (Zimmermann et al., 2019; Balaich et al., 2021).
MAGs allow the prediction of potential functional capacities via analysis
of genome sequences (e.g., by encoding enzymes involved in secondary
metabolite synthesis) and understanding the relationships between
microbiomes and drug metabolism. For example, Maini Rekdal et al.
(2019) identified an inter-species gut bacterial pathway for the meta­
bolism of Levodopa that is the primary medication used Parkinson’s
disease. In this pathway, Levodopa is first converted to dopamine by
Enterococcus faecalis and then converted to m-tyramine by Eggerthella
lenta, thereby reducing the effectiveness of the drug. It should be noted
that not all E.lenta strains convert dopamine to m-tyramine. The strains
capable of this activity have a single-nucleotide polymorphism in dadh
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Microbiological Research 260 (2022) 127023
that converts dopamine to m-tyramine. These results suggest that it is
necessary to analyze the interactions between microorganisms and drug
metabolisms at the strain or even genomic levels. Consequently, evalu­
ating the interactions and mechanisms between MAGs and drug meta­
bolism is of considerable significance for treating diseases and in drug
development.
4.2. Application prospects of MAGs in animals
Animal microbiomes contain abundant biological resources, such as
encoded enzymes (Singh et al., 2014; Rashamuse et al., 2017; Freitas
et al., 2019), antimicrobials (Ochoa et al., 2018; Akbar et al., 2019;
Chevrette et al., 2019) and immunomodulators (Yin et al., 2018). Here,
we focus on the application prospects of MAGs in wild animals, live­
stock, and poultry.
Wild animals exhibit strong immune systems and adaptability, that
may be due to their microbiomes. Levin et al. (2021) discovered new
toxin-metabolizing genes using 5080 MAGs recovered from the gut
metagenomes of over 180 wild animals. These results demonstrate the
potential for using animal metagenome databases to identify microbial
functions and explore biological resources. The study also showed that
the gut microbiome composition of wild animal is correlated with host
heredity, diet, habitat environments, social structures, and lifespans.
Several other studies have also reported similar results (Wu et al., 2018;
Perofsky et al., 2019; Huang et al., 2022). These insights contribute to a
more comprehensive understanding of host-microbiota interactions and
improve strategies for wildlife conservation.
Livestock and poultry such as pigs, cows, sheep and chickens, are
closely related to human life. One study observed significant differences
in gut microbial community composition and function between high and
low feed efficiency host groups (Xie et al., 2021). Consequently,
improving feed efficiency may promote the productivity of livestock and
poultry in addition to food products, such as meat, eggs, milk, and other
products. Feed additives, such as probiotics, prebiotics, and synbiotics,
can improve feed efficiency and animal growth rates, and can also help
to treat diseases such as diarrhea (Markowiak and Slizewska, 2018).
MAGs provide rich resources for identifying potential probiotics sup­
plements for animal feed.
4.3. Application prospects of MAGs for other environments
MAGs have also been recovered from diverse natural environments
(Parks et al., 2017; Nayfach et al., 2020; Danko et al., 2021). Microbial
communities and their metabolites are present in air, building materials,
waters, soils and other environments where they may significant im­
plications for human health (Gilbert and Stephens, 2018; Jack et al.,
2018; Lehtimaki et al., 2021; Zhu et al., 2019). Further, antimicrobial
resistance genes (ARGs) of bacteria can be transferred to humans,
leading to drug ineffectiveness and increasing the risk of disease trans­
mission (Sun et al., 2020; Wu et al., 2022). MAGs in natural environ­
ments are important resources for identifying potential hosts of ARGs,
and facilitate the identification of potential risks to public health in
environments. A recently constructed catalog of global urban microbial
ecosystems and antimicrobial resistance highlighted the potential ap­
plications of MAGs from nature environments in public health (Danko
et al., 2021). Within agriculture, MAGs can allow researchers to explore
the effects of rhizosphere microbiomes and soil microbiomes on nutrient
uptake, disease resistance, stress resistance, and of plant production at
the strain level (Carrion et al., 2019). Further, microorganisms are
important sources of numerous enzymes, antimicrobials, bacteriocins,
and many other natural products, leading to their widespread use in
food, chemical, and pharmaceutical industries and many others (Houde
et al., 2004; Shin et al., 2013; Singh et al., 2016; Hug et al., 2020). Thus,
natural environment MAGs are significant resources for microbiome
research in the fields of public health, agriculture, industry and many
others.
Y. Zhou et al.
5. Challenges and opportunities
The number of MAGs has rapidly increased in recent years with the
development of high-throughput sequencing technologies. However,
uniform bioinformatic procedures and quality standards have yet to be
produced for assembled genomes. Consequently, MAG quality varies
greatly across studies. In addition, important metadata information
related to MAGs, including features, sample sources, and corresponding
sequencing data, are often missing from public records. These lack of
data brings challenges for the reuse or integration of public datasets
(Scholz et al., 2012). The deficiency of this important information also
seriously restricts comparisons among different datasets and in-depth
analysis of microbiome data. Kasmanas et al. (2020) recently con­
structed a curated and standardized database of metadata for human
metagenomes. Further, Liu et al. (2020) provided a practical guide for
the reproducible analysis of microbiome data, suggesting that re­
searchers should submit raw sequence data, detailed metadata, and
associated code with the publication studies. These suggestions provide
a framework for constructing unified metagenomic databases and
standards for metadata submission in the future.
Furthermore, optimized algorithms are required in each step of
generating MAGs from metagenomes to improve assembly efficiency,
MAG quality, and taxonomic resolution. In particular, MAG quality re­
lies on the quality of assembled contigs. However, the correct assembly
of contigs from sequence reads is a challenge due to high strain diversity,
the presence of repeat sequences, and the influence of high abundance
species. Third-generation sequencing technology can produce longer
contigs and more complete genomes (Stewart et al., 2019b), but suffers
from a higher error rate (Jain et al., 2016). Metagenomic assembly from
the combination of both long and short sequence reads can help to
obtain higher quality, or nearly complete genomes, leading to more
accurate taxonomic classification and more comprehensive functional
genome annotation (Bertrand et al., 2019). In contrast to metagenomic
sequencing, single-cell genomic sequencing can produce higher quality
genomes with low abundance, while also linking gene functions to
specific microbial strains. However, challenges exist for single-cell
genomic sequencing via cell-sorting, the presence of chimeric reads
and uneven read coverage (Xu and Zhao, 2018). Consequently, the
combination of metagenomic sequencing and single-cell genomic
sequencing can compensate for individual weaknesses of the two tech­
niques and will help to expand our understanding of the diversity and
functional gene capacity of uncultured bacteria.
The vast majority of MAGs recovered from shotgun metagenomic
sequencing data by metagenomic binning are bacteria, followed by a
small percentage of archaea MAGs. Further, viral sequences are rarely
binned, and are easily combined with bacterial, archaeal, or eukaryotic
sequences (Xie et al., 2021). Thus, archaeal, viral, and fungi MAGs are
lacking. This discrepancy is likely due to variable abundances of mi­
crobial species affecting the assembly of low abundance species. Most
studies that generate MAGs focus on bacterial taxa. Thus, a relatively
adequate understanding of bacterial composition exists for various en­
vironments. However, the interactions between bacterial species remain
unclear. Nevertheless, some investigations of the interactions between
bacteria have been conducted by integrating culturomes, metagenomes
(or 16 S rRNA gene sequencing data), transcriptomes, and mathematical
models (Buffie et al., 2015; Zhao et al., 2018; D’Hoe et al., 2019). Mi­
crobial interactions are critical in structuring the composition and
functional capacity of microbial communities, in addition to regulating
the health and behavior of the hosts. Fungi can affect bacterial growth,
nutrient availability, and ecosystem function (de Menezes et al., 2017;
Pierce et al., 2020). Likewise, phages have been reported to play
essential roles in regulating bacterial diversity and affecting host im­
munity through coevolution with bacteria (Mirzaei and Maurice, 2017;
De Sordi et al., 2019). Moreover, Heyer et al. (2019) showed that mi­
crobial communities in biogas plants are shaped by bacterial-archaeal
syntrophic interactions and phage-bacterial interactions using
9
Microbiological Research 260 (2022) 127023
metaproteomics approaches. Integrating metagenomic, metatran­
scriptomic, metaproteomic, metabolomic and viromic analyses can
provide a systems-level understanding of both the composition and
functional capacities of microbiomes, along with interactions among
microbiota (Bikel et al., 2015; Mallick et al., 2017; Abu-Ali et al., 2018;
Liu et al., 2020). Thus, it is necessary to investigate the causality of
microorganisms in host phenotypes by integrating multiple omics
datasets including transcriptomics, metabolomics, and culturomics.
Assembly-free metagenomic profiling approaches can used to rapidly
obtain genes and functional profiles by aligning metagenomic sequence
reads to reference gene catalogs (Brown et al., 2019). Such approaches
can also help to identify low-abundance species that are difficult to
assemble (Quince et al., 2017). Nevertheless, a comprehensive and
complete catalog of genomes and genes would be required for identi­
fying uncharacterized organisms because it is difficult to identify pre­
viously uncharacterized microorganisms. Consequently, MAG and
non-redundant gene catalog would be valuable resources for
assembly-free metagenomic sequencing data analyses.
6. Conclusions
Genomic binning from metagenomes provides a vast number of un­
cultured microbial genomes from shotgun metagenomic sequencing
data and significantly expands the known phylogenetic diversity of
microbial communities. In this review, we provide a reference for re­
searchers to choose the proper tools and pipelines for metagenomic
sequencing-based studies. Further, the review provides a framework to
better understand the contributions and application prospects of MAGs
in microbiome studies. The quality of draft genomes has yet to be
improved by combining data from multiple sequencing technologies and
improvements in bioinformatics algorithms. Nevertheless, MAGs pro­
vide critical genomic information for isolating and culturing microor­
ganisms, in addition to improving the investigation of relationships
between microbiomes and host phenotypes. Culture-based studies are
consequently critically needed to further elucidate and confirm the
functional capacities of microorganisms related to these MAGs.
Author Contributions
Y. Z. designed, wrote and revised the manuscript. M. L. and J. Y.
edited the manuscript. All authors contributed to the article and
approved the submitted version.
Acknowledgements
We appreciate professor Congying Chen in the State key laboratory
of pig genetic improvement and production technology, Jiangxi Agri­
cultural University for his suggestion on the revision of article. This
work was supported by the National Natural Science Foundation of
China (31772579 and 31760654).
Declaration of Competing Interest
The authors declare that they have no competing interests.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.micres.2022.127023.
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