TAGEDENS E M I N A R S I N P E R I N A T O L O G Y 45 (2021) 151456

Available online at www.sciencedirect.com

Seminars in Perinatology
www.seminperinat.com

Multi ‘omic data integration: A review of concepts,

considerations, and approaches
Tasha M. Santiago-Rodriguez, and Emily B. Hollister*
Diversigen, Inc, 3 Greenway Plaza, Suite 1575, Houston, TX 77046, USA

A R T I C L E I N F O AB STR ACT

Keywords: The application of ‘omic techniques including, but not limited to genomics/metagenomics,
Omics transcriptomics/meta-transcriptomics, proteomics/meta-proteomics, and metabolomics
multi’omic integration to generate multiple datasets from a single sample have facilitated hypothesis generation
metagenomics leading to the identification of biological, molecular and ecological functions and mecha-
metatranscriptomics nisms, as well as associations and correlations. Despite their power and promise, a variety
proteomics of challenges must be considered in the successful design and execution of a multi-omics
metabolomics study. In this review, various ‘omic technologies applicable to single- and meta-organisms
(i.e., host + microbiome) are described, and considerations for sample collection, storage
and processing prior to data generation and analysis, as well as approaches to data storage,
dissemination and analysis are discussed. Finally, case studies are included as examples of
multi-omic applications providing novel insights and a more holistic understanding of bio-
logical processes.
Ó 2021 Elsevier Inc. All rights reserved.

An introduction to ‘omic technologies
‘Omics refers to the comprehensive or global assessment of a
collection of features. An ‘omic-based analysis may include
genomics or metagenomics (i.e., all genes in an organism or
community), transcriptomics or meta-transcriptomics,
metabolomics, proteomics, or other features studied in a
global, typically high-throughput, manner.1 Although reduc-
tionist approaches focused on single genes, proteins, and
pathways have successfully identified key features influenc-
ing health and disease, rarely is a single entity the underlying
cause of a particular phenotype or complex disease. Whereas
analyte by analyte-based approaches were common previ-
ously, many ‘omics platforms have reached a state where
high-throughput, high-resolution, cost-efficient profiling of
samples is the norm. The ability to study information-rich
‘omic data sets has been driven, in large part, by the develop-
ment and increasing availability of array technologies, high-
throughput mass spectrometry and sequencing platforms,
and the development of data and computer science techni-
ques for the movement, management, and integration of
high dimensional data.2
Biological systems rely upon the transfer of information
from nucleic acids to proteins and metabolites in order to
shape function and phenotype. This is referred to as the
‘omics cascade (Fig. 1), and 'omic-driven studies are facilitat-
ing a new, more holistic understanding of systems biology.
This recognizes that many diseases result from complex and/
or heterogeneous processes and a combined ‘lens’ may be
more powerful than single ‘omes alone.3 7 Recent studies
also suggest that outliers in multi-omic data sets may signal
disease progression and could be leveraged as early diagnos-
tic indicators.3,8,9 Such early detection capabilities may help

*Corresponding author.
E-mail address: ehollister@diversigen.com (E.B. Hollister).

https://doi.org/10.1016/j.semperi.2021.151456
0146-0005/Ó 2021 Elsevier Inc. All rights reserved.
2 S E M I N A R S I N P E R I N A T O L O G Y 45 (2021) 151456

Fig. 1 – ‘Omics cascade. Figure depicts depicts the flow of information and various ‘omic technologies used in the characteri-
zation of genome/metagenomes, transcriptome/meta-transcriptomes, proteome/meta-proteomes, and metabolomes in sin-
gle organisms and meta-organisms (i.e., host + microbiome). Figure also shows the utility of each analyte and their half-
lives.

refine approaches for public health screening and diagnosis,
as well as pinpoint and personalize interventions or treat-
ments.10 In the following sections, we present commonly
studied ‘omes, discuss methods and strategies for their anal-
ysis and integration, and highlight multi-omic case studies.
Genomics and metagenomics
Genomics is the characterization of an organism’s entire
genetic content. Typically consisting of DNA (RNA in some
viruses), genomes consist of coding (i.e., genes) and non-coding
regions, and the relative proportions of these vary across the
tree of life. Noncoding regions include introns, regulatory ele-
ments, and repetitive DNA and tend to be more common in
eukaryotes.11 Protein coding sequences represent approxi-
mately 1% of the human genome, but 80-90% of most bacterial
genomes 12. Genomes are characterized through sequencing,
assembly, and comparison with reference genomes. Whole
genome sequencing can be used to identify novel genes or gene
variants, and previously uncharacterized traits may be identi-
fied through genome wide association studies (GWAS), which
link genomic variants with disease states or other phenotypes.
Multiple different approaches exist within genomics. For exam-
ple, exome sequencing is a genome sequencing strategy in
which exons (i.e., protein coding regions) are targeted instead of
whole genomes. Targeted approaches like this can lead to
faster, cheaper, more sensitive gene variant discovery.12
In contrast to single genomes, a metagenome is the collec-
tive genomic content of a community (typically microbial).
The human microbiome is the collection of the microbes that
live in and on the body. Metagenomic studies of the human
microbiome have focused largely on its bacterial members (i.
e., bacteriomics) to date. Despite this, both fungi (mycobiome)
and viruses (virome) are part of the human microbiome.
Although human-associated microbes have been studied for
centuries, interest in the human microbiome has grown
steadily in recent years. Fueled by the recognition that
microbes, both pathogenic and commensal, have the poten-
tial to influence nutrient acquisition, immune and neurologi-
cal development, and long-term health,13 15 as well as
advances in sequencing technology, metagenomic studies of
the human microbiome have become commonplace.
Metagenomic data are generated through untargeted
approaches extracting whole community DNA and using
shotgun sequencing to generate taxonomic and functional
profiles; however, amplicon-based approaches [e.g., 16S ribo-
somal RNA (rRNA) gene or internal transcribed spacer (ITS)
sequencing)] can be used to generate bacterial and archaeal
(16S rRNA gene) or fungal (ITS) taxonomic profiles.16 Genomic
(or metagenomic) data provide information about taxonomy,
phylogenetic relationships, potential function, mutations,
and allelic variations, which can be used to shape hypothe-
ses, guide experiments, and/or suggest mechanisms underly-
ing the appearance or behavior of a system. Although
mutations occur within genomes and metagenomic content
can shift in the context of shifts in community composition,
genomes are typically considered static in the sense that they
do not turn over rapidly and have long half-lives relative to
other ‘omes (e.g., RNA, proteins and metabolites). For
instance, the half-life of DNA in bones is 521 years, but soil
microbial DNA has a half-life varying from 9.1 to 49.7 h.17
 TAGEDENS E M I N A R S I N
Viromics
Viromics refers to the study of viral communities (i.e.,
virome) and may include viruses that infect bacterial,
archaeal, fungal, plant, animal, and/or human cells. Viral
metagenomics typically employs shotgun metagenomic or
meta-transcriptomic sequencing to characterize DNA or RNA
viruses, respectively. Viral metagenomics is challenging
given the small genome and structural sizes of viruses com-
pared to bacteria, their high degree of genome variation (e.g.,
double stranded or single stranded, DNA or RNA-based
genomes), and biases of reference databases toward patho-
gens and well-characterized bacteriophages. These con-
straints often require additional isolation, concentration
steps, and annotation steps relative to general metagenomics
pipelines.18 Despite these challenges, the importance of the
virome in health and diseases is increasingly being discov-
ered using viral metagenomics.18 Viral metagenomics may be
used to detect pathogenic viruses,19 and when paired with
bacterial community profiling correlations between viruses
and bacteria can be used to understand cross-kingdom poten-
tial relationships.20 22 Bacteria-virus interactions have also
been used to understand horizontal gene transfer mediated
by bacteriophages, a process also known as transduction
(and transductomics).23
Transcriptomics and meta-transcriptomics
Transcriptomics is the study of expressed RNA. Transcrip-
tomics often focuses on protein-coding RNA [i.e., messenger
RNA (mRNA)] but can include non-coding RNAs, which coor-
dinate and tune gene expression. Given that expressed genes
represent a fraction of the total genome, transcriptomic sig-
nals can provide focus on the genes and potential mecha-
nisms involved in a biological process of interest.
Transcriptomic profiling can be performed on host tissue or
the microbiome (i.e., meta-transcriptomics), and each pro-
vides different information regarding a system’s biology.
Transcriptomic approaches can be targeted focusing on one
or a few genes, semi-targeted using pre-defined arrays (e.g.,
sequence capture panels and microarrays), or untargeted
using shotgun sequencing.24
Transcriptomic (or meta-transcriptomic) data can be used
to confirm or refute (meta)genomic-based hypotheses. How-
ever, a transcript’s presence does not guarantee translation
into viable protein. RNases are nearly ubiquitously present,
and RNA molecules tend to be highly labile. mRNA half-lives
range from seconds to minutes in well-characterized bacte-
rial and fungal strains, with this range extending to hours for
some human transcripts.25 29 Immediate sample processing
or preservation are crucial for ensuring RNA quantity, quality,
and signal detection.30,31 Adequate depletion of ribosomal
RNA and other non-target molecules are also important for
(meta)transcriptomic data generation.31 Additionally, given
that transcriptomic data reflect responses to conditions pres-
ent at a specific point in time, careful experimentation, sam-
ple handling, and time series analysis are often required.
P E R I N A T O L O G Y 45 (2021) 151456 3
Proteomics and meta-proteomics
Proteomics, the study of expressed proteins, is used to char-
acterize protein identity, abundance, post-translational mod-
ifications, and/or the interactions of proteins (or peptides) in
a system, sample, or matrix. Meta-proteomics characterizes
proteins originating from multiple sources (e.g., host plus
microbiome). Protein abundances are the result of upstream
transcriptional activity,32,33 translational effects, and post-
translational modifications (e.g., phosphorylation, glycosyla-
tion, nitrosylation, and/or ubiquitination), which are difficult
to predict from transcriptional signals alone.34 Proteomic
measurements are typically generated using coupled liquid
chromatography (LC) and mass spectrometry (MS), and they
can be targeted or untargeted. Unlike RNA, proteins are typi-
cally more stable and have longer half-lives (e.g., 10 to 1000 h,
depending on origin).35,36 Proteomics technologies also tend
to be lower-throughput and more expensive in nature.37
Metabolomics
Metabolomics characterizes the small molecules (i.e., metab-
olites) present in a sample or matrix, including amino acids,
fatty acids, carbohydrates, and other compounds. Metabolites
can be derived from host and/or microbiome metabolism,
ingested through diet or medication, and/or environmental
sources and are not limited to a single source (i.e., some
metabolites may be produced by both host and microbe).
Metabolites reflect a variety of upstream biological processes
and can link genotype with phenotype.38 Metabolomic meas-
urements are made using nuclear magnetic resonance (NMR)
or gas or liquid chromatography (GC, LC) paired with mass
spectrometry (GC-MS, LC-MS), and metabolomic studies can
be performed in a targeted or untargeted manner. Metabolite
half-lives vary depending on the metabolite, collection site,
sample matrix, and preservation conditions, with the range
covering hours,39 41 to days.42 Most metabolites are highly
labile, necessitating careful handling and preservation to
maintain signal integrity.43
Epigenomics
Epigenomics addresses genome-wide characterization of
reversible chemical modifications of DNA or DNA-associated
proteins impacting gene expression and regulation.44 Epige-
nomic modifications are associated with several diseases
including Prader-Willi syndrome, Angelman syndrome, and
certain types of cancer.45,46 Epigenomic modifications can
provide information regarding disease status and/or environ-
mental exposure and act as heritable traits. Epigenomic mod-
ifications may be characterized through methylome
sequencing, a modified genomic sequencing approach which
captures methylated genomic regions using restriction
enzymes or bisulfate treatment prior to sequencing. Alterna-
tively, chromatin-immuno-precipitation coupled with
sequencing (ChIP-seq) represents a more targeted approach,
employing antibodies specific to the modification(s) of
4 S E M I N A R S I N P E R I N A T O L O G Y
interest. In both cases, deep sequencing is required to maxi-
mize information gain.
Exposomics
Exposomics is the study of environmental exposures (i.e., the
exposome) and their effects on health and disease. Exposures
may include aspects of the natural (e.g., air, water and soil
quality) and built environments (e.g., quality of workplace and
housing and access to fresh produce), as well as other chemi-
cal exposures and/or pollutants. These factors may exert bio-
logical responses, including inflammation, pro-inflammatory
cytokine secretion, methylation, and gene expression changes,
as well as increased responsiveness to cortisol. These
responses can result in modifications to the microbiome, epi-
genome, and downstream gene expression.47 Exposomics is
frequently linked to other ‘omes, including metabolomics, as
certain molecules may be linked with functional changes asso-
ciated with chronic illness.48
Additional ‘omes
Additional ‘omic fields, many of which are sub-disciplines of
those described above, exist and continue to emerge. Exam-
ples of these include, but are not limited to the lipidome,
inflammasome, and interactome, each of which are described
below.
Lipidomics, a sub-discipline of metabolomics, focuses on
the structure and function of lipids in a cell or organism. Lipi-
domics also assesses interactions among lipids, as well as
between lipids and proteins or metabolites.49 Lipids are
known to influence a number of diseases and their progres-
sion, including cardiovascular disease, obesity, asthma, dia-
betes, and hypertension.49 Lipidomics may complement
metagenomics as a comprehensive analysis of lipid composi-
tion can reveal the current state of a cell’s metabolism. Tech-
niques such as MS, LC-MS, MS imaging, and ion-mobility MS
are often used in lipidomics.49
The inflammasome is defined as the receptors and sensors,
often intracellular proteins, that induce inflammation in
response to pathogens, host protein-derived molecules, and
other stimuli.50 Inflammasome sensors recognize and
respond to lipopolysaccharides, microbial/viral DNA and bac-
terial flagellins, providing protection against microbial infec-
tions.51 Altered inflammasomes are associated with
infection, IBD, cancer, and autoimmune conditions,51 and
studies are increasingly demonstrating associations between
these conditions and the gut microbiota.52,53
The interactome studies protein-protein interactions, as
well as tight complex forming macromolecule-macromole-
cule interactions. This developing field can aid in the discov-
ery of biomarkers and therapeutic approaches.54 One
approach used to study the interactome involves breaking
cells by cryolysis to provide a snapshot of their complexes at
different stages. Analyses are performed as in proteomics
and meta-proteomics usually using MS.55
45 (2021) 151456
Considerations for multi-omic studies
Study design, sample collection and sample storage
Careful planning is required to maximize information gained
and minimize bias in multi-omic studies.56 Well-designed
studies account for the inclusion of sufficient samples for sta-
tistical power, appropriate controls, replication (biological and
technical), collection of enough biomass to support multiple
assays, comprehensive documentation of project metadata
and experimental procedures, and appropriate sample han-
dling. ‘Omic analytes vary with respect to their inherent stabil-
ity, and many require specific collection and preservation
conditions to maintain sample integrity and reliability. This is
particularly important when samples are collected in field-
and community-based settings which preclude immediate
sample processing or -80°C storage. The collection, pre-treat-
ment, or preservation approaches appropriate for some ‘omes
or analytes may not suite others.57 For instance, preservation
of fecal metabolomic samples in 95% ethanol results in strong
concordance with immediate preservation at -80°C, but degra-
dation can occur when 95% ethanol is used for field preserva-
tion of DNA.58 Just as sample collection and preservation are
key considerations, so are methods for analyte extraction and
isolation. Variation among DNA extraction methods can
impact metagenomic profiles,59,60 and protein quantity and
recovery vary according to proteomic extraction method.61
Data resources, storage, and dissemination
Data resources, storage, and dissemination are critical for
multi-omic studies, too. Best practices for data management
and dissemination are evolving and often dictated by funding
agencies, repository owners, and publication requirements.
Data management and dissemination are also shaped by con-
sented use and government-mandated data privacy regula-
tions. Implementing good multi-omic data management
practices is important for data sharing, support of further analy-
ses and new data interpretation, and development of new soft-
ware, tools, and workflows. The FAIR principles (Findability,
Accessibility, Interoperability, and Reusability) have been pro-
posed to guide scientific data management and stewardship.62
Other data standards and guides, including the minimum infor-
mation about any sequence (MIxS) checklists,63 (e.g., MIGS,
MIMS, and MIMARKS) seek to record key information regarding
genomic, metagenomic, and marker gene sequencing projects.
Similarly, MINSEQE (Minimum Information about a high-
throughput nucleotide SEQuencing Experiment; http://fged.org/
projects/minseqe/), MIAME (Minimum Information about a
Microarray Experiment),64 and MIAPE (Minimum Information
About a Proteomics Experiment),65 describe the information
needed to support interpretation and reproducibility for
sequencing, microarray, and proteomics studies, respectively.
Databases
Databases are essential for any ‘omics study, as they aid in
the identification of analytes and provide contextual
 TAGEDENS E M I N A R S I N
information. Although a variety of specialized databases
exist, some of the most frequently used ones are described
below.
Nucleotide and protein sequences
The National Center for Biotechnology Information (NCBI)
houses multiple nucleotide and protein databases, including
GenBank and the Reference Sequence collection (RefSeq).
GenBank is an open access, annotated collection of publicly
deposited nucleotide and protein sequences.66 RefSeq is a
non-redundant version of GenBank. Other repositories
include the Sequence Read Archive (SRA), the European Bioin-
formatics Institute (EMBL-EBI), and the DNA Database of
Japan (DDBJ), each of which contribute to the International
Nucleotide Sequence Database Collaboration (INSDC). The
SRA is specific for sequence data generated on high-through-
put, next-generation sequencing platforms. Specialized data-
bases exist as well, including specific databases for 16S and
18S rRNA gene sequence data, like SILVA,67 the Genome Tax-
onomy Database (GTDB), which has sought to quantitatively
define species via average nucleotide identity (ANI),68 the
MetaHIT (METAgenomics of the Human Intestinal Tract) gene
catalog,69 and the Integrated Microbial Genomes and Micro-
biomes (IMG/M) database.70
Metabolomics
Metabolite information can be accessed through a variety of
repositories. For instance, the Human Metabolome Database
(HMDB) includes predicted MS/MS and GC-MS data, and
metabolite structural information.71 A related database, the
Human Fecal Metabolome database, focuses specifically on
metabolite information from human stool. Each entry con-
tains over 110 data fields that are hyperlinked to other refer-
ence databases, and it allows users to browse concentration
data and associated diseases.72 Similarly, the Metabolomics
Workbench Metabolite Database contains structures and
annotations of biologically relevant metabolites, containing
approximately 136,000 entries collected from public sources
like Chemical Entities of Biological Interest (ChEBI), HMDB,
Biological Magnetic Resonance Bank (BMRB), PubChem, and
the Kyoto Encyclopedia of Genes and Genomes (KEGG).73
METLIN is a resource for metabolite and other molecule data
analysis, representing over 350 chemical classes,74,75 and
MetaboLights covers structures, reference spectra, biological
roles, and data from metabolic experiments.76
Proteins and proteomics
The Universal Protein Resource (UniProt) provides protein
sequence and annotation information, as well as tools includ-
ing local BLAST, alignments, downloadable releases, and data
submission options.77 Other databases aggregate functional
information into functional modules and pathways, includ-
ing KEGG,78 the Clusters of Orthologous Genes (COGs) data-
base,79 and Pfam.80 In addition, multiple easily searchable,
curated databases support proteomics research. Among
these, the PRoteomics IDEntification Database (PRIDE) con-
tains proteomics datasets that are searchable, with a
P E R I N A T O L O G Y 45 (2021) 151456 5
summary containing the description of the project, sample
and processing protocols, as well as contact information.81
Approaches and platforms for multi-omic analysis
and data integration
Despite the increased availability of ‘omic data sets and tools
for their analysis, multi-omic integration remains challeng-
ing.2 Study design, noise, and data interoperability contribute
to this, as do varying definitions of what constitutes a multi-
omic study. “Multi-omic integration” may be used to describe
the analysis of a single 'ome across multiple studies (e.g., a
meta-analysis), as well as the integration of multiple ‘omes
generated on the same set of samples (i.e., “vertical integra-
tion”).82 Similarly, multi-omic integration can be conceptual,
statistical, and/or model-based.57,83 Conceptual integration
combines insights obtained from single ‘omes to form a more
comprehensive understanding of the biology of a system. Sta-
tistical integration focuses on statistical relationships within
and across ‘omes, and model-based integration is the layering
of ‘omic data onto pre-defined system models to understand
molecular organization and function.
In selecting methods for multi-omic data integration, the
nature of one’s data and how it should be handled must be
considered. Many ‘omic data are noisy, sparse (i.e., contain
many zeros), high-dimensional, and contain batch effects.
‘Omic data can be highly heterogeneous, have signals of dif-
fering scale across ‘omes, and vary with respect to being
quantitative, qualitative, and/or compositional.84 These qual-
ities are computationally challenging and often require pre-
processing, including filtering, imputation of missing values,
transformation, normalization, and/or scaling prior to down-
stream analysis. These steps limit impacts of outliers, reduce
the number of features considered, and prevent one ‘ome’s
signals from overwhelming others.85,86 Additionally, spike-in
standards may provide a “ground truth” to facilitate harmoni-
zation across ‘omes.56
Interoperability (i.e., the ability of the data sets to ‘speak’ to
one another) between ‘omic data sets also represents an anal-
ysis obstacle. Although numeric relationships can be consid-
ered without explicit links between 'omes, the ability to
leverage knowledge-based metabolic networks, like those
published by KEGG,78 MetaCyc,87 Reactome,88 and Ingenuity
Pathway Analysis (IPA, QIAGEN Inc., https://digitalinsights.
qiagen.com/products-overview/discovery-insights-portfolio/
analysis-and-visualization/qiagen-ipa/), relies upon one’s
ability to map or overlay data onto pre-existing models and
frameworks. It also requires that identifiers (e.g., gene,
enzyme, compound) be classified using specific ontologies.
Although such models and frameworks are incredibly useful,
it should be noted that they are not comprehensive and may
not fully represent complex systems.
Through conceptual integration, results from one ‘ome are
used to build upon signals observed in another.89 Beyond
seeking consensus (i.e., maximum agreement), studies may
search for complementarity (i.e., useful information found in
each ‘ome but not necessarily shared across them) or lever-
age information contained in one ‘ome to provide context for
another. Consensus-seeking approaches can pinpoint
6 S E M I N A R S I N P E R I N A T O L O G Y
features that contribute to mechanism. Complementarity-
maximizing approaches can provide useful information
about molecular processes and levels at which they are car-
ried out.3 Leveraging ‘omes versus one another is often done
with paired metagenomic and meta-transcriptomic data to
determine transcript to gene ratios. In paired metagenomic/
meta-proteomic studies, metagenome-assembled genes facil-
itate protein identification from MS spectra.90,91
With statistical integration, multiple existing approaches
and models have been applied to ‘omic data sets,82,86,92 and
new algorithms and platforms are introduced regularly. Sta-
tistical integration includes model-based (i.e., statistical mod-
els), multi-variate, and network-based methods, including
many Bayesian approaches. Bayesian approaches leverage a
priori assumptions about the data to calculate posterior prob-
ability distributions, refining our ability to model and predict
outcomes. Bayesian approaches are commonly applied to
high-dimensional data sets, including single and multiple
‘omes, for feature selection. Such approaches seek to identify
features for which support across multiple ‘omes is collec-
tively high, and they are attractive in that they can incorpo-
rate existing biological knowledge.
Statistical integration includes both unsupervised and
supervised analyses. Unsupervised analyses seek to identify
subgroups within a data set, irrespective of known pheno-
types or clinical features. For example, cluster analysis and
Principal coordinates analysis seek to identify groups such
that samples within a group are more like one another than
to those in other groups. Typically, these similarities are
based on a similarity (or distance) metric calculated among
samples in an all versus all manner. Importantly, cluster
number and size are not defined a priori. Clustering can be
used to identify outliers, sub-groups, or assess the distribu-
tion of known sample characteristics across groups.
Network, or association, analysis is another unsupervised
technique used to identify numerical relationships among
‘omic features and samples.84 Analytes and/or samples are
represented as nodes and relationships (i.e., Pearson or
Spearman correlations) are depicted by edges connecting
them. Network analysis can be performed on single or multi-
ple ‘omes. When performed with multiple ‘omes, this is
referred to as concatenation, “early integration”,92 or “single
block” analysis.2. With this approach, all data are combined
into a single matrix prior to downstream analysis, and signal
normalization and scaling are particularly important. Net-
work size, degree of connectivity, and overall topology can be
evaluated, and potential relationships can be inferred from
these results. The numerical relationships identified using
correlation-based approaches may not reflect direct, physical
relationships, nor do they specifically account for complex
interactions.86 Despite the simplicity of these approaches,
association analyses can identify potentially co-regulated
features,93,94 features regulating one another,95 and highlight
dysregulation in the context of disease.82,96
Supervised analyses attempt to model features that can be
used to predict traits (e.g., phenotype or clinical outcome).82
Examples include regression analysis and its multi-variate
extensions, as well as some machine learning algorithms (e.
g., Random Forests). Variable selection (i.e., feature selection)
techniques are an extension of supervised analysis and can
45 (2021) 151456
be achieved using a variety of methods, including partial least
squares regression, canonical correlation analysis (CCA),
sparse CCA, and partial least squares discriminant analysis.
Feature selection can serve as a discovery tool to identify can-
didate biomarkers and may provide mechanistic clues related
to poorly understood phenomena. Variable selection meth-
ods are frequently applied in the analysis of single ‘omic data
sets and may be used as a filtering step preceding the integra-
tion of multiple ‘omes.82 Additional models and techniques
are described in the platforms below and reviewed at length
elsewhere.82,86,92
Model-based integration leverages pre-defined system
models to understand molecular organization and function.
‘Omics data may be pre-processed or pre-filtered (e.g., retain-
ing differentially abundant or expressed features identified
through feature set enrichment analysis or similar
approaches) prior to model mapping, or all data may be
mapped with the intent of identifying reactions and/or path-
ways for which evidence exists. Tools such as the Integrated
Molecular Pathway-Level Analysis (IMPaLA),97 PaintOmics,98
IPA (Qiagen), and the KEGG mapper,78 can be leveraged for
this type of analysis. These approaches are appealing due to
the highly visual nature of their output, and they are useful in
that they can place large amounts of data in biological con-
text. However, as noted above, these models are not fully
comprehensive, may not facilitate the integration of all types
of ‘omic data, and are often limited in their representation of
complex systems.
Software and bioinformatic tools for multi ‘omic
analyses
Multi ‘omic data generation has fueled a need for tools and
platforms to support analysis, integration, and visualization.
A variety of tools have been developed and are becoming
available. Some perform correlation analyses, while others
facilitate covariance-based and/or multiple co-inertia analy-
ses99. Other tools support data visualization, increase
interpretability, and support data sharing and accessibil-
ity.85,100 Several of these tools are described in Table 1, and a
more comprehensive list can be found in https://github.com/
mikelove/awesome-multi-omics. In addition, multiple com-
mercial platforms are available and will likely continue to
emerge (for example, Seven Bridges https://www.seven
bridges.com/)
Case studies and future directions
Multi-omic approaches have been applied in a variety of con-
texts to date, including studies of pregnancy and neonatal
health and disease. As described above, conceptual, statisti-
cal, and model-based integration approaches have been used
to gain insight from layers of ‘omic data. Multi-omic applica-
tions to date have included biomarker discovery, the charac-
terization of microbial dynamics, and host-microbiome
interactions. In most cases, it is anticipated that these discov-
eries will be translated to therapeutics or distilled down to a
 TAGEDENS E M I N A R S I N P E R I N A T O L O G Y 45 (2021) 151456 7

Table 1 – Description of several multi-omics tools for data integration, analysis and/or visualization.

Tool Interface Description Input Supports Supports single Reference

microbiome organisms
97
IMPaLA Web A web interface for the joint List of pathways that con- Yes Yes
analysis of transcriptom- tain at least one gene/pro-
ics or proteomics and tein and/or metabolite
metabolomics data.
Enrichment analysis is
performed with user-
specified lists of metabo-
lites and genes as input.
101
Cystoscape Java A platform for the visualiza- Network file formats Yes Yes
tion of interactions of any
data type through
networks.
98
PaintOmics Web A web tool for the visualiza- Feature quantification file No Yes
tion of multi-omics data- (e.g., proteomics, tran-
sets onto KEGG pathways. scriptomics and
metabolomics)
102
VANTED Java A Java-based framework Network file formats Yes* Yes
which generates network-
based view on large-scale
datasets
103
mixOmics R package An R package which classi- Normalized microarray, Yes Yes*
fies sample groups, iden- mass spectrometry-based
tifies discriminant proteomics, metabolo-
features, and predicts the mics, or sequence count
class of new samples. data (RNA-seq, 16S,
metagenomic)
104
MoCluster R package Applies multiblock multi- Normalized multiple omics Yes* Yes
variate analysis to define datasets
groups of variables that
represent shared patterns
across 'omics datasets. A
clustering algorithm then
aids to discover joint
clusters.
99
Multiple co- R package A multivariate analysis Normalized multiple omics Yes* Yes
inertia analy- method to investigate datasets
sis (mCIA) relationship patterns in
multiple omics datasets.
It utilizes both sparse and
structured sparse
methods.
105
MultiOmics R package A statistical approach for Normalized multiple omics Yes* Yes
factor analy- integrating multi-omics datasets
sis (MOFA) datasets in an unsuper-
vised manner.
106
Pathview Web Web A web-based tool that Gene or compound data in Yes* Yes
maps, integrates, and ren- tab- or comma-delimited
ders biological data to format (txt or csv) with
generate publication- matrix of genes or com-
ready visuals. pounds as rows and sam-
ples as columns.
107
BioMiner Web Integrates various cross- Data import must be per- Yes* Yes
omics high-throughput formed by a dedicated
data sets, focusing on specialist and requires a
cancer and personalized predefined cross-omics
medicine. relationship
108
trackViewer Bioconductor Can be used to visualize Mapped reads, genomic No Yes
package coverage and annotation coverage, SNP informa-
tracks and generate lolli- tion, or methylation sta-
pop and dandelion plots tus of a genomic region
to visualize methylation, converted into Granges
mutation, or variant data. from various file formats.
8 S E M I N A R S I N P E R I N A T O L O G Y 45 (2021) 151456

Table 1 (continued)

Tool Interface Description Input Supports Supports single Reference

microbiome organisms
109
STATegraEMS Web Supports annotation and Data as produced by the Yes* Yes
tracking of analysis pipe- omics equipment.
lines, including RNA-seq,
miRNA-seq, Chip-seq,
Methyl-seq, or Dnase-seq,
as well as proteomics and
metabolomics data.
110
gNOMO Snakemake Integrates and analyzes DNA and RNA paired-end Yes Yes
metagenomics, metatran- reads, as well as MS/MS
scriptomics and metapro- spectra
teomics data from the
microbiome and non-
model host organisms.

* Although the program has been developed specifically for the analysis of single organisms or microbiome data, both analysis types may be supported.

few key analytes for the purposes of diagnosis and/or patient
stratification (Fig. 2).
Multi-omic approaches are being used to understand a vari-
ety of biological phenomena across the lifespan, including
gestational clocks and preterm birth. Preterm birth is a major
cause of neonatal death, and researchers routinely seek to
understand the factors contributing to it. Establishing a thor-
ough understanding of the molecular underpinnings and
chronological changes occurring during term pregnancy may
help identify (molecular) deviations associated with preterm
birth and other pregnancy-related pathologies. Leveraging a
combination of metabolomics, proteomics, transcriptomics,
and statistical models based on “single block” analysis,
Ghaemi et al. found that immune cell signaling responses
model gestational age better than single ‘omes alone.111 Their
results highlight the immunomodulatory capacity of the ste-
roid hormone pregnanolone sulfate as a possible mechanism
for maintaining pregnancy.
In the context of microbiome development in the days and
weeks following birth, another multi-omic analysis used
metagenomics, proteomics, and metabolomics in meconium
and early stool. This study, an example of conceptual integra-
tion, demonstrated transient versus persistent presence of
certain gut microbes, as well as key features reflecting
microbial metabolism.112 Understanding this window of
development is particularly important as the human gut
microbiome develops rapidly following birth and has the
potential to impact health and disease outcomes later in
life.113 Similarly, meta-proteomics has been used to identify
key functional differences in the gut microbiota of preterm
infants.114 As with the preterm birth study described above,
deviations from “healthy” or “normal” progression in micro-
bial community assembly or metabolism may serve as indica-
tors for poor health outcomes and could implicate key
molecular mechanisms associated with them.
Beyond pregnancy and early-life programming, biomarkers
of birthweight, a characteristic having implications for health
later in life, have been identified using correlation analysis of
various ‘omic data sets including DNA methylation profiling,
transcriptomics, inflammation-related proteins, cholesterol,
and anthropometric measurements.5 Abnormal birthweight
is associated with increased mortality, risk of cardiovascular
diseases, mental health problems, and certain cancers later
in life. Recent work has found that both the metabolome and
methylome carry common signatures associated with abnor-
mal birthweight, as well as novel biomarkers, including a
macrophage-derived chemokine. Additionally, cholesterol
levels in cord blood were correlated with birthweight, such

Fig. 2 – Example of multi-omic applications in health and disease. Multi-omic approaches lend themselves to the discovery of
biomarkers and signals contributing to underlying mechanism. Distilled signals based on these discoveries are likely to con-
tribute to the development of therapeutics or become key analytes which serve as the basis for diagnostic tests and/or
patient stratification.
 TAGEDENS E M I N A R S I N
that higher birthweight is associated with increased high-
density lipoprotein cholesterol.5
Another example of network analysis and statistical inte-
gration for biomarker discovery can be found in a study
examining taxonomic, functional, and metabolic differences
in children with inflammatory bowel syndrome (IBS).4 Key
features differentiating cases from controls included micro-
bial species and metabolites like secondary bile acids, sterols,
and steroid-like compounds, and combinations of these sug-
gested promise in their potential to predict case versus con-
trol, an important step forward in diagnosing a condition that
is identified based upon self-reported symptoms.
Moving forward, as the cost of the various ‘omic data sets
described continues to decrease, and more tools are devel-
oped for data integration, storage, sharing, analysis and visu-
alization, we anticipate that the number of studies,
employing multi-omic strategies will continue to grow. Multi-
omic information will aid in the search for markers of health
and disease and help to explain biological phenomena includ-
ing the dynamics of microbial succession and microbe-host
interactions.
Funding
The author(s) received no specific funding for this work.
Disclosure
T.M.S.-R. and E.B.H. are current employees of Diversigen, Inc.
E.B.H. owns OraSure stock/stock options and has received
honoraria for speaking at symposia.
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