10 years of Nature Protocols
The past, present and future of microbiome analyses
Richard Allen White III, Stephen J Callister, Ronald J Moore, Erin S Baker & Janet K Jansson
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
ago and eventually occupied every habitable environment in
2016 Nature America, Inc. All rights reserved.
the planets biosphere. Although microorganisms are known
to be responsible for key functions on Earth, such as carbon
and nutrient cycling, and determining the health and disease
state of the planets plant and animal inhabitants, greater than
99% of the trillions of microbes thought to exist have yet to be
discovered1. In addition, high microbial diversity has made
it difficult to study specific functions carried out by complex
microbial communities in microbiomes (defined as the totality
of microorganisms and their collective genetic material
present in a specific environment such as all microorganisms
inhabiting the soil or human gut)2,3. Fortunately, technological
advances over the last few decades have greatly facilitated
studies of complex microbiomes and their functions. Here
we discuss advances related to nucleic acid sequencing and
mass spectrometry (MS) analyses that have enabled the
exploration and understanding of microbiomes across a range
of environments as well as in our own bodies36.
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Nucleic acid sequencing
Next-generation sequencing. At the forefront of advances in
microbiome research lie the impressive increases in the speed and
throughput of nucleic acid sequencing technologies. In particular,
there has been a revolution in next-generation (NextGen)
sequencing platforms as they have surpassed the traditional
Sanger sequencing method that dominated the field for nearly
three decades (from 1977 to 2005)7. Sequencing a single bacterial
genome using the Sanger dideoxynucleotide-based chain-
termination approach previously was a major endeavor that took
years to complete8,9. The first bacterial genome to be completely
sequenced using the Sanger approach was Haemophilus influenza9
in 1995 (with Escherichia coli10 completed in 1997). Currently,
Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory,
Richland, Washington, USA. Correspondence should be addressed to J.K.J.
(janet.jansson@pnnl.gov) or E.S.B. (erin.baker@pnnl.gov).
Received 8 June; accepted 19 July; published online 29 September 2016;
doi:10.1038/nprot.2016.148
PERSPECTIVE
because of the availability of rapid and inexpensive NextGen
sequencing platforms, it is now possible to sequence complete
bacterial genomes in a matter of hours11.
NextGen sequencing methods have used several different
high-throughput platforms. The first was the Roche GS20 454
sequencer, which was based on the polymerase cleavage of
pyrophosphate, also known as pyrosequencing12,13. Although 454
sequencing was a key technological advance, and 454 sequencers
including the GS20 and GS FLX series machines and reagents
were used for over a decade (approximately 2005 to 2016, http://
www.genomeweb.com/sequencing/roche-shutting-down-454-
sequencing-business), it had several drawbacks including high
cost of sequencing reagents, high homopolymer error rates (i.e.,
errors in reading through the complex repeats), and surface
area loading limitations owing to bead-based DNA molecule
deposition that restrict the throughput and number of reads
obtained. The second NextGen sequencer was the Solexa (now
Illumina) Genome Analyzer (GA), which was introduced in
2006 and incorporated oligonucleotide array flow cells, reversible
chain terminators and bridge PCR reactions14. This technology
is now routinely used to sequence DNA and RNA extracted from
human and environmental microbiomes and can generate >1.8
terabases (TB) of data in a single run. However, the ultimate
goal was to sequence >18,000 human genomes (~3 gigabase-pair
(Gbp) haploid genome) per year at $1,000 per human genome
(http://www.illumina.com/systems/hiseq-x-sequencing-system/
system.html). Illumina currently has several technical platforms
including GA, MiSeq and HiSeq machines, with varying
sequence read lengths (100-300-bp paired-end reads) and
throughputs to try and address this challenge. For example, the
maximum read length with overlapping paired reads on a MiSeq
platform is ~500-550 bp, but that platform has lower throughput
than the HiSeq platform, which generates billions of reads per
run (Fig. 1). A relatively new approach developed by Illumina,
called TruSeq synthetic long reads or Moleculo, results in longer
read lengths (>8 kbp)15, and has facilitated the assembly of highly
complex soil microbiomes16 and other biological samples17,18.
Initial results from these technological advances are enhancing
microbiome assembly into longer contigs1618.
NATURE PROTOCOLS | VOL.11 NO.11 | 2016 | 2049
 PERSPECTIVE
Read length (bp)
1 x 1010 100
1,000
Number of sequences per lane, cell or plate
10,000
1 x 108
Illumina GAIIx (2010)
Illumina GAIIx (2007)
Roche GS FLX+
1 x 106 Roche GS FLX Titanium
Roche GS FLX
Roche GS20
1 x 10 4
2016 Nature America, Inc. All rights reserved.
2004
Figure 1 | Advances in sequencing technologies over the last decade.
Average read length in base pairs is log10-transformed and is the fill
factor of the points on the graph (larger points equal longer read
lengths). Throughput (number of sequence reads) is represented on the
y axis as follows: Roche 454 technology is per plate (i.e., picotiter plate),
Illumina technology is per lane, PacBio and Oxford MinION is per cell.
The year of introduction of each sequencing machine represented is
shown on the x axis.
Single-molecule sequencing technologies. Emerging sequencing
technologies that show great potential for faster and more
informative microbiome studies include single-molecule-based
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DNA sequencers. One example is the single-molecule, real-time
(SMRT) technology from PacBio that relies on tethered DNA
polymerases and zero-mode waveguides to direct light energy
through small volumes of liquids19,20. PacBio currently offers
long DNA sequence reads of ~10-25 kbp and ~300 Mbp per
SMRT cell (Fig. 1; http://www.pacb.com). Because of the use of
tethered polymerases, the PacBio platform can detect unusual
or modified nucleotide bases without chemical modification
during synthesis, such as DNA methylation, owing to the wobble
of the polymerase21. The Oxford Nanopore sequencing platform
is another emerging and promising single-molecule sequencer.
Unlike current platforms, the Oxford platform does not rely on
sequencing by synthesis but instead directly sequences nucleic acid
molecules by threading the strands through a Nanopore22. Oxford
Nanopore can detect DNA modifications like the PacBio platorm,
has an average read length of ~1-2 kbp, and the longest maximum
read length offered by any sequencer (>90 kbp) (Fig. 1)22. The
main benefit of the Oxford Nanopore sequencer is its thumb-drive-
sized format that can be analyzed on a personal labtop computer
in real time using wireless technology (https://nanoporetech.com/
about-us/news).
2050 | VOL.11 NO.11 | 2016 | NATURE PROTOCOLS
Over the last five years, the PacBio platform has become a
robust technology for sequencing microbiome samples for de
novo and metagenomic assembly applications. The caveat is that
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
potentially sequence very large scaffolds (>50 kbp) and increase
genome closures and reconstruction of genomes directly from
Illumina MiSeq v3 environmental samples. A major challenge with these new
technologies, however, is obtaining high-quality and large-
molecular-weight DNA in the range of hundreds of kilobases or
even megabase lengths. Oxford and PacBio platforms are also
still too low-throughput for large-scale studies, but for sequence
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
improvements have enabled many discoveries of the identities
and potential functions of microbes in habitats around the world
and in our own bodies3,4,23,24. Most of the information obtained
about microbiomes has been from the NextGen sequencing
2008 2012 2016 of 16S rRNA genes as a phylogenetic marker for bacteria and
Year archaea. In addition, NextGen sequencing of total community
DNA (i.e., metagenomics) has made it possible to determine
the complement of functional genes associated with specific
microbial groups in diverse environments. For example, some of
our own research has defined the functional gene compositions
in water and sediment samples after the Deepwater Horizon oil
spill in the Gulf of Mexico25,26 and in thawing permafrost27,28.
However, a vast majority of these genes have no known function,
reflecting the immense diversity and biochemical potential of
environmental microbiomes remaining to be discovered.
Metatranscriptomics. Sequencing total mRNA (i.e.,
metatranscriptomics) reveals which genes are expressed
by specific organisms over spatial and temporal scales.
Metatranscriptomics has offered a wealth of knowledge about
the expression of microbial genes in a variety of ecosystems,
including acid-mine drainage29, human gut30, ocean25,26,3134 and
soil27,28. For example, Gilbert et al.34,35, used metatranscriptomics
to determine the seasonal expression patterns of the marine
microbiome in the English Channel. Recently, we used
metatranscriptomics to deduce which organisms identified from
soil genomes were active in soil16. Although Verrucomicrobia
were highly abundant in the soil under investigation, few mRNA
transcripts mapped to their genomes, suggesting that they were
actually transcriptionally dormant. By contrast, the Firmicutes
genomes were found to be transcriptionally active. This revealed
the utility of metatranscriptomics in validating metagenomics
and understanding the relative activities of different members of
microbial communities28.
Mass-spectrometry-based metaproteomic and
metabolomic measurements
Although advances in DNA sequencing have enabled a better
understanding of microbial phylogenetic and functional gene
 PERSPECTIVE
compositions in microbiomes, it is also desirable to know which 800
Instrument resolution
24

proteins (i.e., metaproteomics) and metabolites (i.e., metabolomics) 700 MS/MS (low resolution)

Instrument resolution (x1,000)

20
are produced under specific conditions, and how perturbations 600
MS/MS (high resolution)

Scan rate (spectra/s)

impact microbial functions. The measurement of proteins and 16
500
metabolites produced by microbiomes in different samples has
mainly been achieved using MS. In MS analyses of microbiome 400 12

samples, the biomolecules of interest are ionized in the source, 300

8
separated according to their mass-to-charge ratio in the mass analyzer 200
and finally detected, providing metaproteomic or metabolomic 4
100
measurements with high sensitivity, resolution and throughput.
The introduction of electrospray ionization (ESI) in 1984 0
Q Q FT itrap Ultra ctive itrap Elite ctive sion e HF mos
0
LC LT Q- u
greatly increased the utility of MS measurements for assessment LT
T
Or
Q- TQ-
b
FT
a
lo
b
r Q bit
a
Ex s Or bitrap -Ex rap F xacti ion L
-E
v
Fu
s
u
L L Ve os O O r Q
of biomolecules36. One challenge with ESI is that it is performed l ra p
Ve bit
Or
at atmospheric pressure (760 torr), whereas mass analyzers
2000 2002 2004 2006 2008 2010 2012 2014 2016 Year introduced
normally operate in pressure regimes between 10-6 and 10-11 torr.
This pressure difference accounts for more than nine orders of Figure 2 | The evolution of ion-trap-based MS instruments. Illustration of
magnitude, and if the source and mass analyzer regions are not well how instrument resolution and MS/MS scan rates have increased with time to
provide users with better measurements. However, using the highest values
coupled, huge ion losses occur36,37. Thus, interface designs using ion
for both resolution and MS/MS acquisition is not usually advised for best
funnels and transmission quadrupole regions have been optimized instrument performance. Maximum instrument resolution is shown as red
2016 Nature America, Inc. All rights reserved.

in the last two decades to refocus the ions while continually
decreasing the pressure38. The improvements in these areas resulted
in a decrease in ion losses and higher sensitivity measurements38.
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
challenges required the improvement of the mass analyzer with
respect to resolution, accuracy and MS/MS speed. Quadrupole,
ion trap, time-of-flight and ion cyclotron resonance (ICR) mass
analyzers constituted the majority of mass analyzers used until
the introduction of the orbitrap37 in 2005. Whereas ICR mass
analyzers allowed high-resolution studies in a select number
of laboratories in the 1990s and into the 2000s, the orbitrap
technology made high-resolution capabilities more affordable for
additional laboratories39,40.
Advances in orbitrap technology over the past decade have
npg
bars (values according to left y axis), with MS/MS low-resolution and high-
resolution scan rates indicated by yellow and blue bars (values according
to right y axis), respectively. The instrument and year it was introduced are
shown on the x axis.
from a variety of increasingly complex ecosystems (Fig. 3). For
example, in the early 2000s, Banfield and co-workers were the
first to use a combination of sequencing and MS approaches to
study microbiomes in acid mine drainage with low microbial
diversity41. Subsequently, microbiomes of varying diversity
and complexity, including leaf-cutter ant colonies4244, the
termite gut45, the human gut46, sediments26,47, ocean water
samples25,3135, permafrost soil27,28,48, and native prairie soil16
have been investigated using either advanced sequencing or MS
technologies, or both48 (Fig. 3). As the technologies continue
to improve, we expect information on new and already studied

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.

Microbiomes of increasing complexity: limitations and

future directions 2000 2016

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.

NATURE PROTOCOLS | VOL.11 NO.11 | 2016 | 2051

 PERSPECTIVE
annotation platforms for improving interpretation and multi-
omics integration algorithms. Several other challenges that need
to be addressed for better microbiome analyses include: extraction
of biomolecules from highly diverse and complex sample matrices,
such as soil, sediments and the human gut; assembly of complete
genomes rather than sequence fragments directly from complex
ecosystems; higher speed, throughput and dynamic range of MS
technologies for metaproteomic and metabolomic measurements;
meeting the high computational RAM requirements for de novo
assembly of large metagenomes and metatranscriptomes16;
sufficient storage and analysis options for terabytes to petabytes
of data; and developing statistical and mathematical models to
integrate the data and provide meaningful biological insights.
Challenges aside, the future looks very bright for microbiome
research. Recently, the White House Office of Science and
Technology Policy (OSTP) announced a National Microbiome
Initiative with funding from several federal agencies, industries and
foundations focusing on improved technologies for understanding
microbiomes5,45. We believe this initiative will prompt even faster
2016 Nature America, Inc. All rights reserved.
and more informative nucleic acid sequencing and MS analyses,
in addition to the development of bioinformatics programs that
can quickly analyze microbiomes from a variety of ecosystems.
We also expect the initiative will enable new technologies that
do not currently exist, such as higher-resolution ion-mobility
separations of peptides and metabolites and new databases for
molecular assignments. These technological and computational
improvements will be vital over the next decade for deciphering
the roles of microbes in their natural habitats and determining
the influence of the complex interplay between members of
microbial communities on ecosystem sustainability and health.
Such in-depth knowledge of microbial community phenomes
will facilitate a better understanding of how perturbations such as
climate change and disease affect microbiome functions, enabling
better predictions of the impacts of these changes and facilitating
improved ecosystem sustainability and human health strategies.
npg
ACKNOWLEDGMENTS We thank N. Johnson and C. Brislawn for their assistance in
preparing the figures. This research was supported by the Pan-omics Program that
is funded by the US Department of Energys Office of Biological and Environmental
Research (Genomic Science Program) and the Microbiomes in Transition (MinT)
Laboratory Directed Research and Development Initiative at the Pacific Northwest
National Laboratory. Pacific Northwest National Laboratory is a multi-program
national laboratory operated by Battelle for the Department of Energy under
contract DE-AC06-76RL01830.
AUTHOR CONTRIBUTIONS R.A.W., S.J.C., R.J.M., E.S.B. and J.K.J. all contributed
to this work and commented on the manuscript at all stages.
COMPETING FINANCIAL INTERESTS The authors declare no competing financial
interests.
Reprints and permissions information is available online at http://www.nature.
com/reprints/index.html.
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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.
npg
PERSPECTIVE
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