Over the years, Nature Methods has published many methods to generate and analyze complex sequence data for microbial studies. We cover highlights from our papers below.

Carl Woese set the stage for a molecular taxonomy of microbial life in 1977 by demonstrating that the 16S ribosomal subunit could form the basis of prokaryotic classification. Amplifying markers such as 16S from microbial mixtures really took off with the advent of high-throughput sequencing, which provided a way to rapidly profile communities sampled directly from the environment. Shotgun sequencing approaches are used more and more for taxonomic profiling as well, enabling gene and genomic sequences to be reconstructed for the functional characterization of communities.

Amplicon-based community profiling
The 454 pyrosequencing platform originally dominated efforts to study the 16S locus because of its long sequence reads. In 2008, Rob Knight and colleagues described the use of error-correcting barcodes for pyrosequencing hundreds of samples together.  Then in 2013, Jeffrey Dangl and colleagues took barcoding to a new level by tagging every template molecule during library prep on the Illumina platform in order to remove much of the PCR bias and error introduced during amplification.

On the computational side, Christopher Quince and colleagues presented PyroNoise in 2009 for ‘denoising’ or removing errors from pyrosequencing flowgrams. Jens Reeder and Rob Knight followed a year later with Denoiser, a fast heuristic alternative. Gene Tyson and colleagues moved away from flowgrams with their Acacia software, which corrects sequence files directly and can also work on Ion Torrent data due to its similar error profile containing homopolymeric repeats.

Once cleaned up, marker sequences need to be grouped into ‘operational taxonomic units’ (OTUs) that roughly correspond to genera, species or strains. Among many algorithms that do this, Robert Edgar introduced UPARSE (we realized that there is some ambiguity but it is pronounced YOU-parse) in 2013 for accurate OTU clustering in the face of erroneous or chimeric sequence reads.

To stitch the computational analysis steps together, ‘quantitative insights into microbial ecology’, or QIIME (pronounced chime) from Rob Knight and colleagues offers a user-friendly modular pipeline for amplicon sequence analysis.

Metagenomic community profiling
In shotgun metagenomics approaches, all fragments of genomic DNA in a sample are sequenced and classified. Isidore Rigoutsos and colleagues introduced PhyloPythia in 2007 to assign fragments to higher taxonomic groups or ‘bins’ based on matching the frequency of tetranucleotide sequences with signatures from known taxa. Its faster, open-source successor PhyloPythiaS from Alice McHardy and colleagues came out in 2012.

Arthur Brady and Steven Salzberg also used sequence composition, or combined it with sequence alignment with Phymm and PhymmBL in 2009; their PhymmBL expanded includes additional functionality and parallelization and came out in 2011.

In 2012, Curtis Huttenhower and colleagues described MetaPhlAn, which limits analysis to clade-specific marker genes to speed up the classification of sequence reads. Peer Bork and colleagues also extracted a limited marker set from metagenomic data in their metagenomic OTUs (mOTU) approach in 2013, but used 40 universally conserved prokaryotic genes. Both methods work best in systems like the human gut that have a large number of sequenced reference genomes.

Genomes from mixtures
Earlier this year, Christopher Quince, Anders Andersson and colleagues published an unsupervised binning method called CONCOCT to help reconstruct genomes from mixtures. It uses sequence composition and differential coverage across samples to assign pre-assembled contiguous sequences (contigs) to species or strain bins.

Single-cell sequencing is another way to obtain microbial genomes. Paul Blainey and Stephen Quake discuss challenges and opportunities for single-cell sequencing in a Commentary in our Method of the Year issue in 2014. When cultures are available, long-read single-molecule sequencing technology can provide very high quality genome sequences; the HGAP software from Jonas Korlach and colleagues makes this possible using a single Pacific Biosciences sequencing library.

With genomic sequences in hand, there remains the question of how to fit them within an appropriate taxonomy. Peer Bork and colleagues tackled the problem in 2013 with their species identification (SpecI) tool, that bases classification on the same 40 markers as mOTU.

Functional analysis and ecology
An array of tools have been designed to wrestle ecological and biological insights from metagenomic sequence data, such as the GENE PRediction IMprovement Pipeline (GenePRIMP) for annotating prokaryotic genomes by Amrita Pati and colleagues in 2010 and the metagenomeSeq method to test for the differential microbe abundance across environments or conditions by Mihai Pop and colleagues in 2013 (also see a comment by Bork and colleagues and the authors’ reply).

In 2010, Rob Knight and colleagues compared 51 methods for their ability to identify biologically relevant distribution patterns using real and simulated 16S pyrosequencing data from samples that were clustered or assayed along environmental gradients. In 2012, Jack Gilbert and colleagues developed microbial assemblage prediction (MAP), an artificial neural network approach to model microbial community structure across the Western English Channel that combines time course metagenomic data from a single site with bioclimatic data gathered over the entire channel.

Quality control and bias
Generating accurate and robust microbial sequence data requires rigorous benchmarking and controls, and experimental methods are constantly improving. Nikos Kyrpides and colleagues studied the use of simulated data to evaluate metagenomic analysis methods in 2007. In 2010, Philip Hugenholtz and colleagues evaluated two methods to deplete rRNA from metatranscriptomes.

J Gregory Caporaso and colleagues further demonstrated the effect of Illumina read quality on taxonomic assignment and diversity assessment in 2013, and Scott Kelley and colleagues developed SourceTracker software to identify contaminants in microbial sequencing studies.

We look forward to many more contributions in the field of microbial sequencing.

References:
Alice Carolyn McHardy et al.
Accurate phylogenetic classification of variable-length DNA fragments
Nature Methods 4, 63-72 (2007) doi:10.1038/nmeth976

Konstantinos Mavromatis et al.
Use of simulated data sets to evaluate the fidelity of metagenomic processing methods
Nature Methods, 4 (6), pp. 495-500 (2007) doi:10.1038/nmeth1043

Micah Hamady, Jeffrey J Walker, J Kirk Harris, Nicholas J Gold & Rob Knight
Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex
Nature Methods 5, 235-237 (2008) doi:10.1038/nmeth.1184

Christopher Quince et al.
Accurate determination of microbial diversity from 454 pyrosequencing data
Nature Methods 6, 639-641 (2009) doi:10.1038/nmeth.1361

Arthur Brady & Steven L Salzberg
Phymm and PhymmBL: metagenomic phylogenetic classification with interpolated Markov models
Nature Methods 6, 673-676 (2009) doi:10.1038/nmeth.1358

J Gregory Caporaso et al.
QIIME allows analysis of high-throughput community sequencing data
Nature Methods 7, 335-336 (2010) doi:10.1038/nmeth.f.303

Jens Reeder & Rob Knight
Rapidly denoising pyrosequencing amplicon reads by exploiting rank-abundance distributions
Nature Methods 7, 668-669 (2010) doi:10.1038/nmeth0910-668b

He et al.
Validation of two ribosomal RNA removal methods for microbial metatranscriptomics
Nature Methods 7, 807-812 (2010) doi:10.1038/nmeth.1507

Amrita Pati et al.
GenePRIMP: a gene prediction improvement pipeline for prokaryotic genomes
Nature Methods 7, 455-457 (2010) doi:10.1038/nmeth.1457

Justin Kuczynski,  Zongzhi Liu,  Catherine Lozupone,  Daniel McDonald,  Noah Fierer &  Rob Knight
Microbial community resemblance methods differ in their ability to detect biologically relevant patterns
Nature Methods 7, 813-819 (2010) doi:10.1038/nmeth.1499
Patil et al.
Taxonomic metagenome sequence assignment with structured output models
Nature Methods 8, 191-192 (2011) doi:10.1038/nmeth0311-191

Arthur Brady & Steven L Salzberg
PhymmBL expanded: confidence scores, custom databases, parallelization and more
Nature Methods 8, 367-367 (2011) doi:10.1038/nmeth0511-367

Dan Knights et al.
Bayesian community-wide culture-independent microbial source tracking
Nature Methods 8, 761-763 (2011) doi:10.1038/nmeth.1650

Lauren Bragg, Glenn Stone, Michael Imelfort, Philip Hugenholtz &  Gene W Tyson
Fast, accurate error-correction of amplicon pyrosequences using Acacia
Nature Methods 9, 425-426 (2012) doi:10.1038/nmeth.1990

Nicola Segata et al.
Metagenomic microbial community profiling using unique clade-specific marker genes
Nature Methods 9, 811-814 (2012) doi:10.1038/nmeth.2066

Peter E Larsen,  Dawn Field &  Jack A Gilbert
Predicting bacterial community assemblages using an artificial neural network approach
Nature Methods 9, 621-625 (2012) doi:10.1038/nmeth.1975

Robert C Edgar
UPARSE: highly accurate OTU sequences from microbial amplicon reads
Nature Methods 10, 996-998 (2013) doi:10.1038/nmeth.2604

Derek S Lundberg,  Scott Yourstone,  Piotr Mieczkowski,  Corbin D Jones &  Jeffery L Dangl
Practical innovations for high-throughput amplicon sequencing
Nature Methods 10, 999-1002 (2013) doi:10.1038/nmeth.2634

Shinichi Sunagawa et al.
Metagenomic species profiling using universal phylogenetic marker genes
Nature Methods 10, 1196-1199 (2013) doi:10.1038/nmeth.2693

Daniel R Mende,  Shinichi Sunagawa,  Georg Zeller &  Peer Bork
Accurate and universal delineation of prokaryotic species
Nature Methods 10, 881-884 (2013) doi:10.1038/nmeth.2575

Chen-Shan Chin et al.
Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data
Nature Methods 10, 563-569 (2013) doi:10.1038/nmeth.2474

Nicholas A Bokulich et al.
Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing
Nature Methods 10, 57-59 (2013) doi:10.1038/nmeth.2276

Joseph N Paulson,  O Colin Stine,  Héctor Corrada Bravo &  Mihai Pop
Differential abundance analysis for microbial marker-gene surveys
Nature Methods 10, 1200-1202 (2013) doi:10.1038/nmeth.2658

Paul C Blainey &  Stephen R Quake
Dissecting genomic diversity, one cell at a time
Nature Methods 11, 19-21 (2014) doi:10.1038/nmeth.2783

Johannes Alneberg et al.
Binning metagenomic contigs by coverage and composition
Nature Methods (2014) doi:10.1038/nmeth.3103