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Over the last decade, technical advances in nucleic acid sequencing and mass spectrometry have enabled faster
and more informative metagenomic, metatranscriptomic, metaproteomic and metabolomic measurements. Here we
review key improvements in multi-omic environmental and human microbiome analyses, and discuss developments
required to address current measurement shortcomings.
Microbes evolved on Earth approximately 3.5 billion years because of the availability of rapid and inexpensive NextGen
ago and eventually occupied every habitable environment in sequencing platforms, it is now possible to sequence complete
2016 Nature America, Inc. All rights reserved.
the planets biosphere. Although microorganisms are known bacterial genomes in a matter of hours11.
to be responsible for key functions on Earth, such as carbon NextGen sequencing methods have used several different
and nutrient cycling, and determining the health and disease high-throughput platforms. The first was the Roche GS20 454
state of the planets plant and animal inhabitants, greater than sequencer, which was based on the polymerase cleavage of
99% of the trillions of microbes thought to exist have yet to be pyrophosphate, also known as pyrosequencing12,13. Although 454
discovered1. In addition, high microbial diversity has made sequencing was a key technological advance, and 454 sequencers
it difficult to study specific functions carried out by complex including the GS20 and GS FLX series machines and reagents
microbial communities in microbiomes (defined as the totality were used for over a decade (approximately 2005 to 2016, http://
of microorganisms and their collective genetic material www.genomeweb.com/sequencing/roche-shutting-down-454-
present in a specific environment such as all microorganisms sequencing-business), it had several drawbacks including high
inhabiting the soil or human gut)2,3. Fortunately, technological cost of sequencing reagents, high homopolymer error rates (i.e.,
advances over the last few decades have greatly facilitated errors in reading through the complex repeats), and surface
studies of complex microbiomes and their functions. Here area loading limitations owing to bead-based DNA molecule
we discuss advances related to nucleic acid sequencing and deposition that restrict the throughput and number of reads
mass spectrometry (MS) analyses that have enabled the obtained. The second NextGen sequencer was the Solexa (now
exploration and understanding of microbiomes across a range Illumina) Genome Analyzer (GA), which was introduced in
of environments as well as in our own bodies36.
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Read length (bp) Over the last five years, the PacBio platform has become a
1 x 1010 100 robust technology for sequencing microbiome samples for de
1,000
novo and metagenomic assembly applications. The caveat is that
Number of sequences per lane, cell or plate
10,000
the PacBio platform needs micrograms of high-molecular-weight
DNA (>40 kbp) for library preparation, limiting the samples it
Illumina HiSeq X
Illumina HiSeq v4 can sequence. Oxford Nanopore offers an inexpensive way to
1 x 108 potentially sequence very large scaffolds (>50 kbp) and increase
Illumina GAIIx (2010)
genome closures and reconstruction of genomes directly from
Illumina MiSeq v3 environmental samples. A major challenge with these new
Illumina GAIIx (2007) technologies, however, is obtaining high-quality and large-
molecular-weight DNA in the range of hundreds of kilobases or
Roche GS FLX+ even megabase lengths. Oxford and PacBio platforms are also
1 x 106 Roche GS FLX Titanium
Roche GS FLX
still too low-throughput for large-scale studies, but for sequence
Roche GS20 assembly applications, these single-molecule sequences offer
Illumina Moleculo future promise to the microbiome community.
PacBio RS II
Oxford MinION
Sequencing complex microbiomes. DNA sequencing technology
1 x 10 4
improvements have enabled many discoveries of the identities
and potential functions of microbes in habitats around the world
2016 Nature America, Inc. All rights reserved.
DNA sequencers. One example is the single-molecule, real-time by specific organisms over spatial and temporal scales.
(SMRT) technology from PacBio that relies on tethered DNA Metatranscriptomics has offered a wealth of knowledge about
polymerases and zero-mode waveguides to direct light energy the expression of microbial genes in a variety of ecosystems,
through small volumes of liquids19,20. PacBio currently offers including acid-mine drainage29, human gut30, ocean25,26,3134 and
long DNA sequence reads of ~10-25 kbp and ~300 Mbp per soil27,28. For example, Gilbert et al.34,35, used metatranscriptomics
SMRT cell (Fig. 1; http://www.pacb.com). Because of the use of to determine the seasonal expression patterns of the marine
tethered polymerases, the PacBio platform can detect unusual microbiome in the English Channel. Recently, we used
or modified nucleotide bases without chemical modification metatranscriptomics to deduce which organisms identified from
during synthesis, such as DNA methylation, owing to the wobble soil genomes were active in soil16. Although Verrucomicrobia
of the polymerase21. The Oxford Nanopore sequencing platform were highly abundant in the soil under investigation, few mRNA
is another emerging and promising single-molecule sequencer. transcripts mapped to their genomes, suggesting that they were
Unlike current platforms, the Oxford platform does not rely on actually transcriptionally dormant. By contrast, the Firmicutes
sequencing by synthesis but instead directly sequences nucleic acid genomes were found to be transcriptionally active. This revealed
molecules by threading the strands through a Nanopore22. Oxford the utility of metatranscriptomics in validating metagenomics
Nanopore can detect DNA modifications like the PacBio platorm, and understanding the relative activities of different members of
has an average read length of ~1-2 kbp, and the longest maximum microbial communities28.
read length offered by any sequencer (>90 kbp) (Fig. 1)22. The
main benefit of the Oxford Nanopore sequencer is its thumb-drive- Mass-spectrometry-based metaproteomic and
sized format that can be analyzed on a personal labtop computer metabolomic measurements
in real time using wireless technology (https://nanoporetech.com/ Although advances in DNA sequencing have enabled a better
about-us/news). understanding of microbial phylogenetic and functional gene
proteins (i.e., metaproteomics) and metabolites (i.e., metabolomics) 700 MS/MS (low resolution)
in the last two decades to refocus the ions while continually bars (values according to left y axis), with MS/MS low-resolution and high-
decreasing the pressure38. The improvements in these areas resulted resolution scan rates indicated by yellow and blue bars (values according
in a decrease in ion losses and higher sensitivity measurements38. to right y axis), respectively. The instrument and year it was introduced are
shown on the x axis.
However, analyzing biomolecules in complex microbiomes
by MS is still very difficult owing to the high dynamic range
and thousands of components present in a given sample. These from a variety of increasingly complex ecosystems (Fig. 3). For
challenges required the improvement of the mass analyzer with example, in the early 2000s, Banfield and co-workers were the
respect to resolution, accuracy and MS/MS speed. Quadrupole, first to use a combination of sequencing and MS approaches to
ion trap, time-of-flight and ion cyclotron resonance (ICR) mass study microbiomes in acid mine drainage with low microbial
analyzers constituted the majority of mass analyzers used until diversity41. Subsequently, microbiomes of varying diversity
the introduction of the orbitrap37 in 2005. Whereas ICR mass and complexity, including leaf-cutter ant colonies4244, the
analyzers allowed high-resolution studies in a select number termite gut45, the human gut46, sediments26,47, ocean water
of laboratories in the 1990s and into the 2000s, the orbitrap samples25,3135, permafrost soil27,28,48, and native prairie soil16
technology made high-resolution capabilities more affordable for have been investigated using either advanced sequencing or MS
additional laboratories39,40. technologies, or both48 (Fig. 3). As the technologies continue
Advances in orbitrap technology over the past decade have to improve, we expect information on new and already studied
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included higher-resolution measurements and faster MS/MS microbial communities to multiply, providing greater insight into
scan rates in both the linear ion trap (low resolution) and orbitrap microbial community phenotypes, or phenomes49.
(high resolution). A plot of the maximum resolution and MS/ There are still many challenges that need to be addressed in
MS scan speeds for MS trapping instruments introduced over order to gain a deeper understanding of the molecular functions
the last 15 years (Fig. 2), reveals that both of these features have of microbiomes5,49. One of the biggest obstacles in microbial
been optimized to obtain the best possible biomolecule coverage community analyses are the bioinformatics and computational
and accuracy in each measurement. However, owing to the bottlenecks. Examples of these include building gene catalogs
complexity of microbiome samples, additional technologies such to ameliorate reads and peptide assignments, biome-specific
as one-dimensional and two-dimensional liquid chromatography
separations and gas-phase ion mobility spectrometry are also Microbiome complexity and multi-omics analysis timeline
being used to increase the number of proteins and metabolites
Extreme Termite Soil and
identified. These separation technologies reduce the complexity of environments hindgut Ocean sediment
the sample before detection, allowing less suppression in the ion Leaf cutter
Human gut Permafrost
ant colony
trap and detector owing to the many molecules present in each
microbiome sample, and enabling higher coverage of the proteins
and metabolites in a given sample39,40.
Developments in nucleic acid sequencing and MS technologies Figure 3 | Approximate timeline with examples of increasingly complex
over the last decade have made it possible to analyze microbiomes microbiomes analyzed by sequencing and/or other omics technologies.
ACKNOWLEDGMENTS We thank N. Johnson and C. Brislawn for their assistance in response to Deepwater Horizon oil spill. ISME J. 8, 14641475 (2014).
preparing the figures. This research was supported by the Pan-omics Program that 27. Mackelprang, R. et al. Metagenomic analysis of a permafrost microbial
is funded by the US Department of Energys Office of Biological and Environmental community reveals a rapid response to thaw. Nature 480, 368371 (2011).
Research (Genomic Science Program) and the Microbiomes in Transition (MinT) 28. Hultman, J. et al. Multi-omics of permafrost, active layer and thermokarst
Laboratory Directed Research and Development Initiative at the Pacific Northwest bog soil microbiomes. Nature 521, 208212 (2015).
National Laboratory. Pacific Northwest National Laboratory is a multi-program 29. Goltsman, D.S.A., Comolli, L.R., Thomas, B.C. & Banfield, J.F. Community
national laboratory operated by Battelle for the Department of Energy under transcriptomics reveals unexpected high microbial diversity in acidophilic
contract DE-AC06-76RL01830. biofilm communities. ISME J. 9, 10141023 (2015).
30. Zoetendal, E.G. et al. The human small intestinal microbiota is driven by rapid
AUTHOR CONTRIBUTIONS R.A.W., S.J.C., R.J.M., E.S.B. and J.K.J. all contributed uptake and conversion of simple carbohydrates. ISME J. 6, 14151426 (2012).
to this work and commented on the manuscript at all stages. 31. Shi, Y., Tyson, G.W., Eppley, J.M. & DeLong, E.F. Integrated
metatranscriptomic and metagenomic analyses of stratified microbial
assemblages in the open ocean. ISME J. 5, 9991013 (2011).
COMPETING FINANCIAL INTERESTS The authors declare no competing financial
32. Frias-Lopez, J. et al. Microbial community gene expression in ocean surface
interests.
waters. Proc. Natl. Acad. Sci. USA 105, 38053810 (2008).
33. Shi, Y., Tyson, G.W. & DeLong, E.F. Metatranscriptomics reveals unique microbial
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small RNAs in the oceans water column. Nature 459, 266269 (2009).
com/reprints/index.html.
34. Gilbert, J.A. et al. Detection of large numbers of novel sequences in the
metatranscriptomes of complex marine microbial communities. PLoS One 3,
1. Locey, K.J. & Lennon, J.T. Scaling laws predict global microbial diversity. e3042 (2008).
Proc. Natl. Acad. Sci. USA 113, 59705975 (2016). 35. Gilbert, J.A. et al. Metagenomes and metatranscriptomes from the L4 long-
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EDITOR SUMMARY
In this Perspective, Janet Jansson and colleagues review the development of microbiome analysis technologies over the past decade, and
comment on the future potential of this fast-moving field.
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