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. agpandCvcmuy2acaped22oThabhk2onmCcain mately 3 5 billio the planet's biosphere.Although microorganisms are known bacterial genomes in amatter of hours rth.such as carbor state of the planet's plant and animal inhabitants.greater than pyrophosphate,also known as pyrosequencing nhigh microb micoomnmicrobomes(efathe totaity of microorganisms and their collective genetic material ts.hi 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 )Genome Analyzer (GA),which ofenvironments as well as in our own bodie Nucleic acid sequencing DNA nd RNA ing At the forefront of advances in he pe tion (NextGer Gbp)haploid me ear at $00 peh equencing platforms as they have surpa sed the traditional (http://www.il mina.com/systems/hiseq equencing-system/ nated the fe eral technical ome using the Sanger dideoxynucleotide-based chain (100300-bp paired-end reads)and approach previously endeavor that took throughputs to try and address this challenge.For exampethe in(wthcompeted n9).Current P than the hisea platform which generates billions of reads per o,res NATURE PR0T0C0LS|70L.11N0.11|2016|2049NATURE PROTOCOLS | VOL.11 NO.11 | 2016 | 2049 10 years of Nature Protocols 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 contigs16–18. Microbes evolved on Earth approximately 3.5 billion years ago and eventually occupied every habitable environment in the planet’s 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 planet’s 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 bodies3–6. 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, 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. 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 npg © 2016 Nature America, Inc. All rights reserved