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10 years of Nature Protocols PERSPECTIVE

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 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 planet’s 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 planet’s 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 bodies3–6.
npg

2006 and incorporated oligonucleotide array flow cells, reversible
chain terminators and bridge PCR reactions14. This technology
Nucleic acid sequencing is now routinely used to sequence DNA and RNA extracted from
Next-generation sequencing. At the forefront of advances in human and environmental microbiomes and can generate >1.8
microbiome research lie the impressive increases in the speed and terabases (TB) of data in a single run. However, the ultimate
throughput of nucleic acid sequencing technologies. In particular, goal was to sequence >18,000 human genomes (~3 gigabase-pair
there has been a revolution in next-generation (NextGen) (Gbp) haploid genome) per year at $1,000 per human genome
sequencing platforms as they have surpassed the traditional (http://www.illumina.com/systems/hiseq-x-sequencing-system/
Sanger sequencing method that dominated the field for nearly system.html). Illumina currently has several technical platforms
three decades (from 1977 to 2005)7. Sequencing a single bacterial including GA, MiSeq and HiSeq machines, with varying
genome using the Sanger dideoxynucleotide-based chain- sequence read lengths (100-300-bp paired-end reads) and
termination approach previously was a major endeavor that took throughputs to try and address this challenge. For example, the
years to complete8,9. The first bacterial genome to be completely maximum read length with overlapping paired reads on a MiSeq
sequenced using the Sanger approach was Haemophilus influenza9 platform is ~500-550 bp, but that platform has lower throughput
in 1995 (with Escherichia coli10 completed in 1997). Currently, than the HiSeq platform, which generates billions of reads per
run (Fig. 1). A relatively new approach developed by Illumina,
Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, called TruSeq synthetic long reads or Moleculo, results in longer
Richland, Washington, USA. Correspondence should be addressed to J.K.J. read lengths (>8 kbp)15, and has facilitated the assembly of highly
(janet.jansson@pnnl.gov) or E.S.B. (erin.baker@pnnl.gov).
complex soil microbiomes16 and other biological samples17,18.
Received 8 June; accepted 19 July; published online 29 September 2016; Initial results from these technological advances are enhancing
doi:10.1038/nprot.2016.148 microbiome assembly into longer contigs16–18.

NATURE PROTOCOLS | VOL.11 NO.11 | 2016 | 2049

. PacBio currently offers including acid-mine drainage29. suggesting that they were Unlike current platforms.23.e.000 novo and metagenomic assembly applications. transcripts mapped to their genomes. informative microbiome studies include single-molecule-based metatranscriptomics) reveals which genes are expressed npg DNA sequencers. Average read length in base pairs is log10-transformed and is the fill the complement of functional genes associated with specific factor of the points on the graph (larger points equal longer read microbial groups in diverse environments. understanding of microbial phylogenetic and functional gene 2050 | VOL. few mRNA is another emerging and promising single-molecule sequencer. Oxford the utility of metatranscriptomics in validating metagenomics Nanopore can detect DNA modifications like the PacBio platorm..31–34 and long DNA sequence reads of ~10-25 kbp and ~300 Mbp per soil27. For example.com/ Although advances in DNA sequencing have enabled a better about-us/news). PERSPECTIVE Read length (bp) Over the last five years.28. Throughput (number of sequence reads) is represented on the our own research has defined the functional gene compositions y axis as follows: Roche 454 technology is per plate (i. Gilbert et al. a vast majority of these genes have no known function. By contrast.20. cell or plate 10. Although Verrucomicrobia of the polymerase21. metagenomics) has made it possible to determine Figure 1 | Advances in sequencing technologies over the last decade.34. 1.pacb. we used or modified nucleotide bases without chemical modification metatranscriptomics to deduce which organisms identified from during synthesis. Sequencing total mRNA (i. reflecting the immense diversity and biochemical potential of environmental microbiomes remaining to be discovered. 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.24. but for sequence Roche GS20 assembly applications. The caveat is that Number of sequences per lane. Oxford and PacBio platforms are also 1 x 106 Roche GS FLX Titanium Roche GS FLX still too low-throughput for large-scale studies. picotiter plate). read length offered by any sequencer (>90 kbp) (Fig.11 NO. The Oxford Nanopore sequencing platform were highly abundant in the soil under investigation. and in our own bodies3. and understanding the relative activities of different members of has an average read length of ~1-2 kbp. Inc. some of lengths). Emerging sequencing technologies that show great potential for faster and more Metatranscriptomics. (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. limiting the samples it Illumina HiSeq X Illumina HiSeq v4 can sequence. however..11 | 2016 | NATURE PROTOCOLS . and the longest maximum microbial communities28. For example. human gut30. In addition. Recently. the Firmicutes sequencing by synthesis but instead directly sequences nucleic acid genomes were found to be transcriptionally active. the PacBio platform has become a 1 x 1010 100 robust technology for sequencing microbiome samples for de 1. the Oxford platform does not rely on actually transcriptionally dormant.26. A major challenge with these new Illumina GAIIx (2007) technologies. Because of the use of to determine the seasonal expression patterns of the marine tethered polymerases.28.000 the PacBio platform needs micrograms of high-molecular-weight DNA (>40 kbp) for library preparation. real-time by specific organisms over spatial and temporal scales. owing to the wobble soil genomes were active in soil16. 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). 1)22. 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. However. NextGen sequencing of total community DNA (i.26 and in thawing permafrost27.35. in water and sediment samples after the Deepwater Horizon oil Illumina technology is per lane. PacBio RS II Oxford MinION Sequencing complex microbiomes. http://www.e. used metatranscriptomics SMRT cell (Fig. such as DNA methylation. is obtaining high-quality and large- molecular-weight DNA in the range of hundreds of kilobases or Roche GS FLX+ even megabase lengths. spill in the Gulf of Mexico25. the PacBio platform can detect unusual microbiome in the English Channel. Single-molecule sequencing technologies. PacBio and Oxford MinION is per cell. The main benefit of the Oxford Nanopore sequencer is its thumb-drive.e. through small volumes of liquids19.4. Most of the information obtained about microbiomes has been from the NextGen sequencing 2004 2008 2012 2016 of 16S rRNA genes as a phylogenetic marker for bacteria and Year archaea. these single-molecule sequences offer Illumina Moleculo future promise to the microbiome community. All rights reserved. One example is the single-molecule. The year of introduction of each sequencing machine represented is shown on the x axis. This revealed molecules by threading the strands through a Nanopore22. ocean25.

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. The measurement of proteins and 16 500 metabolites produced by microbiomes in different samples has mainly been achieved using MS. or phenomes49. additional technologies such to ameliorate reads and peptide assignments. 300 8 separated according to their mass-to-charge ratio in the mass analyzer 200 and finally detected. metaproteomics) and metabolites (i. 3). we expect information on new and already studied npg included higher-resolution measurements and faster MS/MS microbial communities to multiply. or both48 (Fig.31–35. 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. permafrost soil27. Illustration of magnitude. However. 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. to right y axis).40. 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. 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. PERSPECTIVE compositions in microbiomes. providing metaproteomic or metabolomic 4 100 measurements with high sensitivity. respectively. reveals that both of these features have of microbiomes5.11 NO. As the technologies continue Advances in orbitrap technology over the past decade have to improve.000) 20 are produced under specific conditions. technologies.48. NATURE PROTOCOLS | VOL.11 | 2016 | 2051 . the analyzers allowed high-resolution studies in a select number termite gut45. (high resolution).. Thus. sediments26.47. Examples of these include building gene catalogs complexity of microbiome samples.49. The instrument and year it was introduced are shown on the x axis. and enabling higher coverage of the proteins and metabolites in a given sample39. For challenges required the improvement of the mass analyzer with example. microbiomes of varying diversity the introduction of the orbitrap37 in 2005. 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. 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. However. 3). and native prairie soil16 technology made high-resolution capabilities more affordable for have been investigated using either advanced sequencing or MS additional laboratories39. the orbitrap samples25. Whereas ICR mass and complexity. using the highest values coupled. Subsequently. the biomolecules of interest are ionized in the source. accuracy and MS/MS speed. owing to the bottlenecks. first to use a combination of sequencing and MS approaches to ion trap. These from a variety of increasingly complex ecosystems (Fig. However. Quadrupole. ocean water of laboratories in the 1990s and into the 2000s. with MS/MS low-resolution and high- decreasing the pressure38. u greatly increased the utility of MS measurements for assessment LT T Or Q. in the early 2000s. Inc. it is also desirable to know which 800 Instrument resolution 24 proteins (i. 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. providing greater insight into scan rates in both the linear ion trap (low resolution) and orbitrap microbial community phenotypes.. the human gut46. 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. and how perturbations 600 MS/MS (high resolution) Scan rate (spectra/s) impact microbial functions.28. huge ion losses occur36. 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. including leaf-cutter ant colonies42–44. resolution and throughput. In MS analyses of microbiome 400 12 samples. This pressure difference accounts for more than nine orders of Figure 2 | The evolution of ion-trap-based MS instruments. metabolomics) 700 MS/MS (low resolution) Instrument resolution (x1. 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. One challenge with ESI is that it is performed l ra p Ve bit Or at atmospheric pressure (760 torr). All rights reserved. Maximum instrument resolution is shown as red © 2016 Nature America. in the last two decades to refocus the ions while continually bars (values according to left y axis).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 biomolecules36.37.e. 2).e. Banfield and co-workers were the respect to resolution.40. 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.

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