Characterization of the Gut Microbiome Using 16S or Shotgun Metagenomics
Juan Jovel, Jordan Patterson, Weiwei Wang, Naomi Hotte, Sandra O'Keefe, Troy Mitchel, Troy Perry, Dina Kao, Andrew L Mason, Karen L Madsen, Gane K-S Wong, Juan Jovel, Jordan Patterson, Weiwei Wang, Naomi Hotte, Sandra O'Keefe, Troy Mitchel, Troy Perry, Dina Kao, Andrew L Mason, Karen L Madsen, Gane K-S Wong
Abstract
The advent of next generation sequencing (NGS) has enabled investigations of the gut microbiome with unprecedented resolution and throughput. This has stimulated the development of sophisticated bioinformatics tools to analyze the massive amounts of data generated. Researchers therefore need a clear understanding of the key concepts required for the design, execution and interpretation of NGS experiments on microbiomes. We conducted a literature review and used our own data to determine which approaches work best. The two main approaches for analyzing the microbiome, 16S ribosomal RNA (rRNA) gene amplicons and shotgun metagenomics, are illustrated with analyses of libraries designed to highlight their strengths and weaknesses. Several methods for taxonomic classification of bacterial sequences are discussed. We present simulations to assess the number of sequences that are required to perform reliable appraisals of bacterial community structure. To the extent that fluctuations in the diversity of gut bacterial populations correlate with health and disease, we emphasize various techniques for the analysis of bacterial communities within samples (α-diversity) and between samples (β-diversity). Finally, we demonstrate techniques to infer the metabolic capabilities of a bacteria community from these 16S and shotgun data.
Keywords: 16S rRNA gene sequencing; bioinformatics; diversity analysis; functional profiling; gut microbiome; shotgun metagenomics; taxonomic classification.
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References
- Abubucker S., Segata N., Goll J., Schubert A. M., Izard J., Cantarel B. L., et al. . (2012). Metabolic reconstruction for metagenomic data and its application to the human microbiome. PLoS Comput. Biol. 8:e1002358. 10.1371/journal.pcbi.1002358
- Aho A., Hopcroft J., Ullman J. (1973). On finding lowest common ancestors in trees, in Proc. 5th ACM Symp. Theory of Computing (STOC), (New York, NY: ACM; ), 253–265.
- Antharam V. C., Li E. C., Ishmael A., Sharma A., Mai V., Rand K. H., et al. . (2013). Intestinal dysbiosis and depletion of butyrogenic bacteria in Clostridium difficile infection and nosocomial diarrhea. J. Clin. Microbiol. 51, 2884–2892. 10.1128/JCM.00845-13
- Aronesty E. (2011). Command-Line Tools for Processing Biological Sequencing Data ea-utils. Expression Analysis. Durham, NC: Available online at:
- Arslan N. (2014). Obesity, fatty liver disease and intestinal microbiota. World J. Gastroenterol. 20, 16452–16463. 10.3748/wjg.v20.i44.16452
- Bajaj J. S., Betrapally N. S., Hylemon P. B., Heuman D. M., Daita K., White M. B., et al. . (2015). Salivary microbiota reflects changes in gut microbiota in cirrhosis with hepatic encephalopathy. Hepatology 62, 1260–1271. 10.1002/hep.27819
- Barlow G. M., Yu A., Mathur R. (2015). Role of the gut microbiome in obesity and diabetes mellitus. Nutr. Clin. Pract. 30, 787–797. 10.1177/0884533615609896
- Beals E. (1984). Bray-Curtis ordination: an effective strategy for analysis of multivariate ecological data. Adv. Ecol. Res. 14, 1–55.
- Bhattacharjee S., Lukiw W. J. (2013). Alzheimer's disease and the microbiome. Front. Cell. Neurosci. 7:153 10.3389/fncel.2013.00153
- Boisvert S., Raymond F., Godzaridis E., Laviolette F., Corbeil J. (2012). Ray Meta: scalable de novo metagenome assembly and profiling. Genome Biol. 13:R122. 10.1186/gb-2012-13-12-r122
- Bolhuis H., Cretoiu M. S., Stal L. J. (2014). Molecular ecology of microbial mats. FEMS Microbiol. Ecol. 90, 335–350. 10.1111/1574-6941.12408
- Brady A., Salzberg S. (2011). PhymmBL expanded: confidence scores, custom databases, parallelization and more. Nat. Methods 8:367. 10.1038/nmeth0511-367
- Brady A., Salzberg S. L. (2009). Phymm and PhymmBL: metagenomic phylogenetic classification with interpolated Markov models. Nat. Methods 6, 673–676. 10.1038/nmeth.1358
- Bray J. R., Curtis J. T. (1957). An ordination of upland forest communities of southern Wisconsin. Ecol. Monogr. 27, 325–349.
- Brestoff J. R., Artis D. (2013). Commensal bacteria at the interface of host metabolism and the immune system. Nat. Immunol. 4, 676–684. 10.1038/ni.2640
- Broderick N. A. (2015). A common origin for immunity and digestion. Front. Immunol. 6:72. 10.3389/fmicb.2015.00531
- Brown J., de Vos W. M., DiStefano P. S., Doré J., Huttenhower C., Knight R., et al. . (2013). Translating the human microbiome. Nat. Biotechnol. 31, 304–308. 10.1038/nbt.2543
- Buttigieg P. L., Ramette A. (2014). A guide to statistical analysis in microbial ecology: a community-focused, living review of multivariate data analyses. FEMS Microbiol. Ecol. 90, 543–550. 10.1111/1574-6941.12437
- Caporaso J. G., Kuczynski J., Stombaugh J., Bittinger K., Bushman F. D., Costello E. K., et al. . (2010). QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7, 335–336. 10.1038/nmeth.f.303
- Caporaso J. G., Lauber C. L., Walters W. A., Berg-Lyons D., Lozupone C. A., Turnbaugh P. J., et al. . (2011). Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc. Natl. Acad. Sci. U.S.A. 108, 4516–4522. 10.1073/pnas.1000080107
- Chakravorty S., Helb D., Burday M., Connell N., Alland D. (2007). A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria. J. Microbiol. Methods 69, 330–339. 10.1016/j.mimet.2007.02.005
- Chang J. Y., Antonopoulos D. A., Kalra A., Tonelli A., Khalife W. T., Schmidt T. M., et al. . (2008). Decreased diversity of the fecal Microbiome in recurrent Clostridium difficile-associated diarrhea. J. Infect. Dis. 197, 435–438. 10.1086/525047
- Chen W., Zhang C. K., Cheng Y., Zhang S., Zhao H. (2013). A comparison of methods for clustering 16S rRNA sequences into OTUs. PLoS ONE 8:e70837. 10.1371/journal.pone.0070837
- C. Human Microbiome Project R. (2012a). A framework for human microbiome research. Nature 486, 215–221. 10.1038/nature11209
- C. Human Microbiome Project R. (2012b). Structure, function and diversity of the healthy human microbiome. Nature 486, 207–214. 10.1038/nature11234
- Cole J. R., Wang Q., Fish J. A., Chai B., McGarrell D. M., Sun Y., et al. . (2014). Ribosomal Database Project: data and tools for high throughput rRNA analysis. Nucleic Acids Res. 42, D633–D642. 10.1093/nar/gkt1244
- Darling A. E., Jospin G., Lowe E., Matsen F. A. IV, Eisen, J. A. (2014). PhyloSift: phylogenetic analysis of genomes and metagenomes. Peer J. 2:e243. 10.7717/peerj.243
- Dash S., Clarke G., Berk M., Jacka F. N. (2015). The gut microbiome and diet in psychiatry: focus on depression. Curr. Opin. Psychiatry 28, 1–6. 10.1097/YCO.0000000000000117
- DeSantis T. Z., Hugenholtz P., Larsen N., Rojas M., Brodie E. L., Keller K., et al. (2006). Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARApplied, B., and environmental. Microbiology 72, 5069–5072. 10.1128/AEM.03006-05
- Dillies M. A., Rau A., Aubert J., Hennequet-Antier C., Jeanmougin M., Servant N., et al. . (2013). A comprehensive evaluation of normalization methods for Illumina high-throughput RNA sequencing data analysis. Brief Bioinformat. 14, 671–683. 10.1093/bib/bbs046
- Dinan T. G., Borre Y. E., Cryan J. F. (2014). Genomics of schizophrenia: time to consider the gut microbiome? Mol. Psychiatry 19, 1252–1257. 10.1038/mp.2014.93
- Edgar R. C. (2010). Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26, 2460–2461. 10.1093/bioinformatics/btq461
- Edgar R. C. (2013). UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat. Methods 10, 996–998. 10.1038/nmeth.2604
- Efron B., Stein C. (1981). The jackknife estimate of variance. Ann. Statist. 9, 586–596.
- Eren A. M., Maignien L., Sul W. J., Murphy L. G., Grim S. L., Morrison H. G., et al. . (2013). Oligotyping: differentiating between closely related microbial taxa using 16S rRNA gene data. Methods Ecol. Evol. 4, 1111–1119. 10.1111/2041-210X.12114
- Eren A. M., Morrison H. G., Lescault P. J., Reveillaud J., Vineis J. H., Sogin M. L. (2014). Minimum entropy decomposition: unsupervised oligotyping for sensitive partitioning of high-throughput marker gene sequences. ISME J. 9, 968–979. 10.1038/ismej.2014.195
- Faith J. J., Guruge J. L., Charbonneau M., Subramanian S., Seedorf H., Goodman A. L., et al. . (2013). The long-term stability of the human gut microbiota. Science 341:1237439. 10.1126/science.1237439
- Flint H. J., Scott K. P., Louis P., Duncan S. H. (2012). The role of the gut microbiota in nutrition and health. Nat. Rev. Gastroenterol. Hepatol. 9, 577–589. 10.1038/nrgastro.2012.156
- Forster S. C., Browne H. P., Kumar N., Hunt M., Denise H., Mitchell A., et al. . (2016). HPMCD: the database of human microbial communities from metagenomic datasets and microbial reference genomes. Nucleic Acids Res. 44, D604–D609. 10.1093/nar/gkv1216
- Franzosa E. A., Hsu T., Sirota-Madi A., Shafquat A., Abu-Ali G., Morgan X. C., et al. . (2015). Sequencing and beyond: integrating molecular ‘omics’ for microbial community profiling. Nat. Rev. Microbiol. 13, 360–372. 10.1038/nrmicro3451
- Franzosa E. A., Morgan X. C., Segata N., Waldron L., Reyes J., Earl A. M., et al. . (2014). Relating the metatranscriptome and metagenome of the human gut. Proc. Natl. Acad. Sci. U.S.A. 111, E2329–E2338. 10.1073/pnas.1319284111
- Garrett W. S., Gordon J. I., Glimcher L. H. (2010). Homeostasis and inflammation in the intestine. Cell 140, 859–870. 10.1053/j.gastro.2011.02.047
- Gevers D., Pop M., Schloss P. D., Huttenhower C. (2012). Bioinformatics for the Human Microbiome Project. PLoS Comput. Biol. 8:e1002779. 10.1371/journal.pcbi.1002779
- Greenblum S., Turnbaugh P. J., Borenstein E. (2012). Metagenomics systems biology of the human gut microbiome reveals topological shifts associated with obesity and inflammatory bowel disease. Proc. Natl. Acad. Sci. U.S.A. 109, 594–599. 10.1073/pnas.1116053109
- Hartstra A. V., Bouter K. E., Bäckhed F., Nieuwdorp M. (2015). Insights into the role of the microbiome in obesity and type 2 diabetes. Diabetes Care 38, 159–165. 10.2337/dc14-0769
- Heinken A., Thiele I. (2015). Systems biology of host-microbe metabolomics. Wiley interdisciplinary reviews. Syst. Biol. Med. 7, 195–219. 10.1002/wsbm.1301
- Huson D. H., Mitra S., Ruscheweyh H. J., Weber N., Schuster S. C. (2011). Integrative analysis of environmental sequences using MEGAN4. Genome Res. 21, 1552–1560. 10.1101/gr.120618.111
- Huttenhower C., Knight R., Brown C. T., Caporaso J. G., Clemente J. C., Gevers D., et al. . (2014a). Advancing the microbiome research community. Cell 159, 227–230. 10.1016/j.cell.2014.09.022
- Huttenhower C., Kostic A. D., Xavier R. J. (2014b). Inflammatory bowel disease as a model for translating the microbiome. Immunity 40, 843–854. 10.1016/j.immuni.2014.05.013
- Janda J. M., Abbott S. L. (2007). 16S rRNA gene sequencing for bacterial identification in the diagnostic laboratory: pluses, perils, and pitfalls. J. Clin. Microbiol. 45, 2761–2764. 10.1128/JCM.01228-07
- Joice R., Yasuda K., Shafquat A., Morgan X. C., Huttenhower C. (2014). Determining microbial products and identifying molecular targets in the human microbiome. Cell Metab. 20, 731–741. 10.1016/j.cmet.2014.10.003
- Joshi N. A., Fass J. N. (2011). Sickle: A Sliding-Window, Adaptive, Quality-Based Trimming Tool for FastQ files. [Software] Version 1.33. Available online at:
- Kaminski J., Gibson M. K., Franzosa E. A., Segata N., Dantas G., Huttenhower C. (2015). High-specificity targeted functional profiling in microbial communities with ShortBRED. PLoS Comput. Biol. 11:e1004557. 10.1371/journal.pcbi.1004557
- Kanehisa M., Goto S., Sato Y., Kawashima M., Furumichi M., Tanabe M. (2004). Data, information, knowledge and principle: back to metabolism in KEGG. Nucleic Acids Res. 42, D199–D205. 10.1093/nar/gkt1076
- Kielbasa S. M., Wan R., Sato K., Horton P., Frith M. C. (2011). Adaptive seeds tame genomic sequence comparison. Genome Res. 21, 487–493. 10.1101/gr.113985.110
- Knight R., Jansson J., Field D., Fierer N., Desai N., Fuhrman J. A., et al. . (2012). Unlocking the potential of metagenomics through replicated experimental design. Nat. Biotechnol. 30, 513–520. 10.1038/nbt.2235
- Knights D., Lassen K. G., Xavier R. J. (2013). Advances in inflammatory bowel disease pathogenesis: linking host genetics and the microbiome. Gut 62, 1505–1510. 10.1136/gutjnl-2012-303954
- Kostic A. D., Xavier R. J., Gevers D. (2014). The microbiome in inflammatory bowel disease: current status and the future ahead. Gastroenterology 146, 1489–1499. 10.1053/j.gastro.2014.02.009
- Kristiansson E., Hugenholtz P., Dalevi D. (2009). ShotgunFunctionalizeR: an R-package for functional comparison of metagenomes. Bioinformatics 25, 2737–2738. 10.1093/bioinformatics/btp508
- Kuczynski J., Liu Z., Lozupone C., McDonald D., Fierer N., Knight R. (2010). Microbial community resemblance methods differ in their ability to detect biologically relevant patterns. Nat. Methods 7, 813–819. 10.1038/nmeth.1499
- Kultima J. R., Sunagawa S., Li J., Chen W., Chen H., Mende D. R., et al. . (2012). MOCAT: a metagenomics assembly and gene prediction toolkit. PLoS ONE 7:e47656. 10.1371/journal.pone.0047656
- Langille M. G., Zaneveld J., Caporaso J. G., McDonald D., Knights D., Reyes J. A., et al. . (2013). Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences. Nat. Biotechnol. 31, 814–821. 10.1038/nbt.2676
- Laurence M., Hatzis C., Brash D. E. (2014). Common contaminants in Next-Generation Sequencing that hinder discovery of low-abundance microbes. PLoS ONE 9:e97876. 10.1371/journal.pone.0097876
- Leprieur F., Albouy C., De Bortoli J., Cowman P. F., Bellwood D. R., Mouillot D. (2012). Quantifying phylogenetic beta diversity: distinguishing between ‘true’ turnover of lineages and phylogenetic diversity gradients. PLoS ONE 7:e42760. 10.1371/journal.pone.0042760
- Levy R., Borenstein E. (2014). Metagenomic systems biology and metabolic modeling of the human microbiome: from species composition to community assembly rules. Gut Microbes 5, 265–270. 10.4161/gmic.28261
- Ley R. E., Peterson D. A., Gordon J. I. (2006). Ecological and evolutionary forces shaping microbial diversity in the human intestine. Cell 124, 837–848. 10.1016/j.cell.2006.02.017
- Liu Z., DeSantis T. Z., Andersen G. L., Knight R. (2008). Accurate taxonomy assignments from 16S rRNA sequences produced by highly parallel pyrosequencers. Nucleic Acids Res. 36, e120. 10.1093/nar/gkn491
- Lozupone C. A., Knight R. (2008). Species divergence and the measurement of microbial diversity. FEMS Microbiol. Rev. 32, 557–578. 10.1111/j.1574-6976.2008.00111.x
- Lozupone C. A., Stombaugh J., Gonzalez A., Ackermann G., Wendel D., Vázquez-Baeza Y., et al. . (2013). Meta-analyses of studies of the human microbiota. Genome Res. 23, 1704–1714. 10.1101/gr.151803.112
- Lozupone C., Hamady M., Knight R. (2006). UniFrac–an online tool for comparing microbial community diversity in a phylogenetic context. BMC Bioinformatics 7:371. 10.1186/1471-2105-7-371
- Lozupone C., Knight R. (2005). UniFrac: a new phylogenetic method for comparing microbial communities. Appl. Environ. Microbiol. 71, 8228–8235. 10.1128/AEM.71.12.8228-8235.2005
- Mardis E. R. (2008). Next-generation DNA sequencing methods. Annu. Rev. Genomics Hum. Genet. 9, 387–402. 10.1146/annurev.genom.9.081307.164359
- Markowitz V. M., Ivanova N. N., Szeto E., Palaniappan K., Chu K., Dalevi D., et al. . (2008). IMG/M: a data management and analysis system for metagenomes. Nucleic Acids Res. 36, D534–D538. 10.1093/nar/gkm869
- Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 17 10.14806/ej.17.1.200 Available online at: .
- Martín R., Miquel S., Langella P., Bermudez-Humaran L. G. (2014). The role of metagenomics in understanding the human microbiome in health and disease. Virulence 5, 413–423. 10.4161/viru.27864
- Martínez I., Wallace G., Zhang C., Legge R., Benson A. K., Carr T. P., et al. . (2009). Diet-induced metabolic improvements in a hamster model of hypercholesterolemia are strongly linked to alterations of the gut microbiota. Appl. Environ. Microbiol. 75, 4175–4184. 10.1128/AEM.00380-09
- Mende D. R., Sunagawa S., Zeller G., Bork P. (2013). Accurate and universal delineation of prokaryotic species. Nat. Methods 10, 881–884. 10.1038/nmeth.2575
- Meyer F., Paarmann D., D'souza M., Olson R., Glass E. M., Kubal M., et al. . (2008). The metagenomics RAST server - a public resource for the automatic phylogenetic and functional analysis of metagenomes. BMC Bioinformatics 9:386. 10.1186/1471-2105-9-386
- Mikheenko A., Saveliev V., Gurevich A. (2015). MetaQUAST: evaluation of metagenome assemblies. Bioinformatics 32, 1088–1090. 10.1093/bioinformatics/btv697
- Mitra S., Rupek P., Richter D., Urich T., Gilbert J. A., Meyer F., et al. . (2011). Functional analysis of metagenomes and metatranscriptomes using SEED and KEGG. BMC Bioinformatics 12:S21. 10.1186/1471-2105-12-S1-S21
- Monte L., Ghelardi R. J. (1964). A table for calculating the equitability component of species diversity. J. Anim. Ecol. 33, 217–225.
- Morgan X. C., Huttenhower C. (2014). Meta'omic analytic techniques for studying the intestinal microbiome. Gastroenterology 146, 1437–1448 e1. 10.1053/j.gastro.2014.01.049
- Namiki T., Hachiya T., Tanaka H., Sakakibara Y. (2012). MetaVelvet: an extension of Velvet assembler to de novo metagenome assembly from short sequence reads. Nucleic Acids Res. 40, e155. 10.1093/nar/gks678
- Navas-Molina J. A., Peralta-Sánchez J. M., González A., McMurdie P. J., Vázquez-Baeza Y., Xu Z., et al. . (2013). Advancing our understanding of the human microbiome using QIIME. Meth. Enzymol. 531, 371–444. 10.1016/B978-0-12-407863-5.00019-8
- Ni J., Yan Q., Yu Y. (2013). How much metagenomic sequencing is enough to achieve a given goal? Sci. Rep. 3, 1–7. 10.1038/srep01968
- Nicholson J. K., Holmes E., Kinross J., Burcelin R., Gibson G., Jia W., et al. . (2012). Host-gut microbiota metabolic interactions. Science 336, 1262–1267. 10.1126/science.1223813
- Nielsen B., Gürakan G. C., Unlu G. (2014). Kefir: a multifaceted fermented dairy product. Probiot. Antimicrob. Proteins 6, 123–135. 10.1007/s12602-014-9168-0
- Norman J. M., Handley S. A., Baldridge M. T., Droit L., Liu C. Y., Keller B. C., et al. . (2015). Disease-specific alterations in the enteric virome in inflammatory bowel disease. Cell 160, 447–460. 10.1016/j.cell.2015.01.002
- Norman J. M., Handley S. A., Virgin H. W. (2014). Kingdom-agnostic metagenomics and the importance of complete characterization of enteric microbial communities. Gastroenterology 146, 1459–1469. 10.1053/j.gastro.2014.02.001
- Novais R. C., Thorstenson Y. R. (2011). The evolution of Pyrosequencing(R) for microbiology: from genes to genomes. J. Microbiol. Methods 86, 1–7. 10.1016/j.mimet.2011.04.006
- Oksanen J., Blanchet F., Kindt R., Legendre P., Minchin P., O'Hara R., et al. (2015). Vegan Community Ecology Package. R package version 2.2-1. Available online at:
- Ounit R., Wanamaker S., Close T. J., Lonardi S. (2015). CLARK: fast and accurate classification of metagenomic and genomic sequences using discriminative k-mers. BMC Genomics 16:236. 10.1186/s12864-015-1419-2
- Overbeek R., Begley T., Butler R. M., Choudhuri J. V., Chuang H. Y., Cohoon M., et al. . (2005). The subsystems approach to genome annotation and its use in the project to annotate 1000 genomes. Nucleic Acids Res. 33, 5691–5702. 10.1093/nar/gki866
- Paulson J. N., Stine O. C., Bravo H. C., Pop M. (2013). Differential abundance analysis for microbial marker-gene surveys. Nat. Methods 10, 1200–1202. 10.1038/nmeth.2658
- Pimentel M., Mathur R., Chang C. (2013). Gas and the microbiome. Curr. Gastroenterol. Rep. 15, 356. 10.1007/s11894-013-0356-y
- Qichao T., Zhili H., Jizhong Z. (2014). Strain/species identification in metagenomes using genome-specific markers. Nucleic Acids Res. 42, e67. 10.1093/nar/gku138
- Qin J., Li R., Raes J., Arumugam M., Burgdorf K. S., Manichanh C., et al. . (2010). A human gut microbial gene catalogue established by metagenomic sequencing. Nature 464, 59–65. 10.1038/nature08821
- Qin J., Li Y., Cai Z., Li S., Zhu J., Zhang F., et al. . (2012). A metagenome-wide association study of gut microbiota in type 2 diabetes. Nature 490, 55–60. 10.1038/nature11450
- Quast C., Pruesse E., Yilmaz P., Gerken J., Schweer T., Yarza P., et al. . (2013). The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 41, D590–D596. 10.1093/nar/gks1219
- Quinn G. P., Keough M. J. (2002). Experimental Design and Data Analysis for Biologists. Cambridge: Cambridge University Press.
- Ramette A. (2007). Multivariate analyses in microbial ecology. FEMS Microbiol. Ecol. 62 142–160. 10.1111/j.1574-6941.2007.00375.x
- Reyes A., Haynes M., Hanson N., Angly F. E., Heath A. C., Rohwer F., et al. . (2010). Viruses in the faecal microbiota of monozygotic twins and their mothers. Nature 466, 334–338. 10.1038/nature09199
- Riesenfeld C. S., Schloss P. D., Handelsman J. (2004). Metagenomics: genomic analysis of microbial communities. Annu. Rev. Genet. 38, 525–552. 10.1146/annurev.genet.38.072902.091216
- Rinke C., Schwientek P., Sczyrba A., Ivanova N., Anderson I., Cheng J., et al. . (2013). Insights into the phylogeny and coding potential of microbial dark matter. Nature 499, 431–437. 10.1038/nature12352
- Ritari J., Salojärvi J., Lahti L., de Vos W. M. (2015). Improved taxonomic assignment of human intestinal 16S rRNA sequences by a dedicated reference database. BMC Genomics 16:1056. 10.1186/s12864-015-2265-y
- Rokach L., Maimon O. (2005). Clustering Methods. Data Mining and Knowledge Discovery Handbook. New York, NY: Springer.
- Salter S. J., Cox M. J., Turek E. M., Calus S. T., Cookson W. O., Moffatt M. F., et al. . (2014). Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biol. 12:87. 10.1186/s12915-014-0087-z
- Schaeffer L., Pimentel H., Bray N., Melsted P., Pachter L. (2015). Pseudoalignment for metagenomic read assignment. arXiv 1510.07371.
- Schaubeck M., Clavel T., Calasan J., Lagkouvardos I., Haange S. B., Jehmlich N., et al. . (2015). Dysbiotic gut microbiota causes transmissible Crohn's disease-like ileitis independent of failure in antimicrobial defence. Gut 65, 225–237. 10.1136/gutjnl-2015-309333
- Schloss P. D., Handelsman J. (2008). A statistical toolbox for metagenomics: assessing functional diversity in microbial communities. BMC Bioinformatics 9:34. 10.1186/1471-2105-9-34
- Schloss P. D., Westcott S. L. (2011). Assessing and improving methods used in operational taxonomic unit-based approaches for 16S rRNA gene sequence analysis. Appl. Environ. Microbiol. 10, 3219–3226. 10.1128/AEM.02810-10
- Schloss P. D., Westcott S. L., Ryabin T., Hall J. R., Hartmann M., Hollister E. B., et al. . (2009). Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl. Environ. Microbiol. 75, 7537–7541. 10.1128/AEM.01541-09
- Segata N., Boernigen D., Tickle T. L., Morgan X. C., Garrett W. S., Huttenhower C. (2013). Computational meta'omics for microbial community studies. Mol. Syst. Biol. 9, 666 10.1038/msb.2013.22
- Segata N., Waldron L., Ballarini A., Narasimhan V., Jousson O., Huttenhower C. (2012). Metagenomic microbial community profiling using unique clade-specific marker genes. Nat. Methods 9, 811–814. 10.1038/nmeth.2066
- Shannon C. E. (1948). A mathematical theory of communication. Bell Syst. Techn. J. 27, 379–423.
- Soergel D. A., Dey N., Knight R., Brenner S. E. (2012). Selection of primers for optimal taxonomic classification of environmental 16S rRNA gene sequences. ISME J. 6, 1440–1444. 10.1038/ismej.2011.208
- Stackebrandt E., Ebers J. (2006). Molecular taxonomic parameters: tarnished gold standards. Microbiol. Today 33, 152–155. 10.1038/msb.2013.22
- Strong M. J., Xu G., Morici L., Splinter Bon-Durant S., Baddoo M., Lin Z., et al. . (2014). Microbial contamination in next generation sequencing: implications for sequence-based analysis of clinical samples. PLoS Pathog. 10:e1004437. 10.1371/journal.ppat.1004437
- Sun Y., Cai Y., Huse S. M., Knight R., Farmerie W. G., Wang X., et al. . (2012). A large-scale benchmark study of existing algorithms for taxonomy-independent microbial community analysis. Brief. Bioinformatics 13, 107–121. 10.1093/bib/bbr009
- Tikhonov M., Leach R. W., Wingreen N. (2015). Interpreting 16S metagenomic data without clustering to achieve sub-OTU resolution. ISME J. 9, 68–80. 10.1038/ismej.2014.117
- Treangen T. J., Koren S., Sommer D. D., Liu B., Astrovskaya I., Ondov B., et al. . (2013). MetAMOS: a modular and open source metagenomic assembly and analysis pipeline. Genome Biol. 14, R2. 10.1186/gb-2013-14-1-r2
- Tuomisto H. (2010). A diversity of beta diversities: straightening up a concept gone awry. Part 1. Defining beta diversity as a function of alpha and gamma diversity. Ecography 33, 2–22. 10.1111/j.1600-0587.2009.05880.x
- Turnbaugh P. J., Gordon J. I. (2009). The core gut microbiome, energy balance and obesity. J. Physiol. 587, 4153–4158. 10.1113/jphysiol.2009.174136
- Turnbaugh P. J., Hamady M., Yatsunenko T., Cantarel B. L., Duncan A., Ley R. E., et al. . (2009). A core gut microbiome in obese and lean twins. Nature 457, 480–484. 10.1038/nature07540
- Vetrovský T., Baldrian P. (2013). The variability of the 16S rRNA gene in bacterial genomes and its consequences for bacterial community analyses. PLoS ONE 8:e57923. 10.1371/journal.pone.0057923
- Vincent C., Stephens D. A., Loo V. G., Edens T. J., Behr M. A., Dewar K., et al. . (2013). Reductions in intestinal Clostridiales precede the development of nosocomial Clostridium difficile infection. Microbiome 1, 18. 10.1186/2049-2618-1-18
- Vital M., Howe A. C., Tiedje J. M. (2014). Revealing the bacterial butyrate synthesis pathways by analyzing (meta)genomic data. MBio 5, e00889. 10.1128/mBio.00889-14
- Waldor M. K., Tyson G., Borenstein E., Ochman H., Moeller A., Finlay B. B., et al. . (2015). Where next for microbiome research? PLoS Biol. 13:e1002050. 10.1371/journal.pbio.1002050
- Wang J., Shen J., Wu Y., Tu C., Soininen J., Stegen J. C., et al. . (2013). Phylogenetic beta diversity in bacterial assemblages across ecosystems: deterministic versus stochastic processes. ISME J. 7, 1310–1321. 10.1038/ismej.2013.30
- Wang Q., Garrity G. M., Tiedje J. M., Cole J. R. (2007). Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ. Microbiol. 73, 5261–5267. 10.1128/AEM.00062-07
- Wang W., Jovel J., Halloran B., Wine E., Patterson J., Ford G., et al. . (2015). Metagenomic analysis of microbiome in colon tissue from subjects with inflammatory bowel diseases reveals interplay of viruses and bacteria. Inflamm. Bowel Dis. 21, 1419–1427. 10.1097/MIB.0000000000000344
- Weiss S., Amir A., Hyde E. R., Metcalf J. L., Song S. J., Knight R. (2014). Tracking down the sources of experimental contamination in microbiome studies. Genome Biol. 15, 564. 10.1186/s13059-014-0564-2
- Whittaker R. H. (1972). Evolution and measurement of species diversity. Taxon 21, 213–251.
- Wood D. E., Salzberg S. L. (2014). Kraken: ultrafast metagenomic sequence classification using exact alignments. Genome Biol. 15:R46. 10.1186/gb-2014-15-3-r46
- Wu M., Eisen J. A. (2008). A simple, fast, and accurate method of phylogenomic inference. Genome Biol. 9:R151. 10.1186/gb-2008-9-10-r151
- Yen S., McDonald J. A., Schroeter K., Oliphant K., Sokolenko S., Blondeel E. J., et al. . (2015). Metabolomic analysis of human fecal microbiota: a comparison of feces-derived communities and defined mixed communities. J. Proteome Res. 14, 1472–1482. 10.1021/pr5011247
- Yoon S. S., Kim E. K., Lee W. J. (2015). Functional genomic and metagenomic approaches to understanding gut microbiota-animal mutualism. Curr. Opin. Microbiol. 24C, 38–46. 10.1016/j.mib.2015.01.007
- Zhu A., Sunagawa S., Mende D. R., Bork P. (2015). Inter-individual differences in the gene content of human gut bacterial species. Genome Biol. 16, 82. 10.1186/s13059-015-0646-9
- Zur E. F., Ieno E. N., Smith G. M. (2007). Analyzing Ecological Data. New York, NY: Springer.
Source: PubMed