Getting Started with Microbiome Analysis: Sample Acquisition to Bioinformatics

Ranjit Kumar1, Peter Eipers2, Rebecca B. Little3, Michael Crowley4, David K. Crossman4, Elliot J. Lefkowitz5, Casey D. Morrow2

1 Center for Clinical and Translational Sciences, University of Alabama at Birmingham, 2 Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, 3 Department of Nutrition Sciences, University of Alabama at Birmingham, 4 Department of Genetics and Heflin Center for Genomic Science, University of Alabama at Birmingham, 5 Department of Microbiology, University of Alabama at Birmingham
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 18.8
DOI:  10.1002/0471142905.hg1808s82
Online Posting Date:  July, 2014
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Abstract

Historically, in order to study microbes, it was necessary to grow them in the laboratory. It was clear though that many microbe communities were refractory to study because none of the members could be grown outside of their native habitat. The development of culture‐independent methods to study microbiota using high‐throughput sequencing of the 16S ribosomal RNA gene variable regions present in all prokaryotic organisms has provided new opportunities to investigate complex microbial communities. In this unit, the process for a microbiome analysis is described. Many of the components required for this process may already exist. A pipeline is described for acquisition of samples from different sites on the human body, isolation of microbial DNA, and DNA sequencing using the Illumina MiSeq sequencing platform. Finally, a new analytical workflow for basic bioinformatics data analysis, QWRAP, is described, which can be used by clinical and basic science investigators. Curr. Protoc. Hum. Genet. 82:18.8.1‐18.8.29. © 2014 by John Wiley & Sons, Inc.

Keywords: microbe communities; 16S rRNA genes; microbiome analysis pipeline; QWRAP bioinformatics analysis

     
 
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Table of Contents

  • Introduction
  • Acquisition of Samples for Microbiome Analysis
  • Basic Protocol 1: Oral Sample Acquisition: Saliva
  • Alternate Protocol 1: Oral Sample Acquisition: Buccal
  • Basic Protocol 2: Acquisition of Vaginal and Skin Samples
  • Basic Protocol 3: Acquisition of Fecal Samples Using Pre‐Moistened Wipe Method
  • Alternate Protocol 2: Acquisition of Fecal Samples Using the Stool Method
  • Support Protocol 1: Transporting Fecal Samples to the Laboratory
  • Support Protocol 2: Processing of Fecal Samples after Shipment
  • Multiplexed 16S Amplicon Sequencing on the MiSeq System
  • Basic Protocol 4: DNA Isolation
  • Basic Protocol 5: Generate Amplicon Library by PCR
  • Basic Protocol 6: Sample Preparation for Loading onto the Illumina MiSeq
  • Support Protocol 3: Quantification of PCR DNA Using Pico Green
  • Microbiome Data Analysis
  • Basic Protocol 7: Installing QWRAP and Other Dependencies
  • Basic Protocol 8: Microbiome Analysis Using QWRAP
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Oral Sample Acquisition: Saliva

  Materials
  • 50‐ml conical collection tube

Alternate Protocol 1: Oral Sample Acquisition: Buccal

  Materials
  • Any type of cotton‐tip collection swab (e.g., Fisher Scientific, cat. no. 1490710)

Basic Protocol 2: Acquisition of Vaginal and Skin Samples

Basic Protocol 3: Acquisition of Fecal Samples Using Pre‐Moistened Wipe Method

  Materials
  • Scott Naturals moist wipe
  • Biohazard laboratory bags
  • Small or medium Therapak box (if shipping, available from Fisher Scientific) or another container with a biohazard bag
  • FedEx Clinical Pak (a bag overwrap for Clinical Sample shipment)

Alternate Protocol 2: Acquisition of Fecal Samples Using the Stool Method

  Materials
  • Modified Cary Blair medium (see recipe)
  • Commode collection pan (i.e., plastic hat or toilet hat)
  • Plastic spoons
  • Gloves
  • 120‐ml urine collection vial
  • Small or medium Therapak box (Fisher Scientific)
  • FedEx ClinicalPak
  • 2‐ or 2.5‐gallon plastic zipper bag or garbage bag to use as disposal bag for collection pan
  • 3‐oz. PolarPack freezer gel pack, optional

Support Protocol 1: Transporting Fecal Samples to the Laboratory

Support Protocol 2: Processing of Fecal Samples after Shipment

Basic Protocol 4: DNA Isolation

  Materials
  • Fecal DNA Isolation Kit (Zymo Research, cat. no. D6010)
  • Microbiome sample (see Basic Protocols protocol 11 to protocol 43 and Alternate Protocols protocol 21 and protocol 52)
  • Tris‐EDTA buffer, optional
  • Microspectrometer (http://www.nanodrop.com/)

Basic Protocol 5: Generate Amplicon Library by PCR

  Materials
  • LongAmp Taq PCR kit (NEB, cat. no. E5200S)
  • Degenerate PCR primers specific for the V4 region (Eurofins—mwg/operon (http://www.operon.com)
  • Template DNA (see protocol 8)
  • 1.0% agarose/Tris‐borate‐EDTA gel
  • QIAquick Gel Extraction Kit (Qiagen, cat. no. 28704)
  • Thermal cycler
  • Electrophoresis apparatus and power source
  • UV illuminator and gel photography equipment

Basic Protocol 6: Sample Preparation for Loading onto the Illumina MiSeq

  Materials
  • PCR sample (see protocol 9)
  • Tris·Cl, pH 8.5/Tween 20 (10 mM/0.1%; see appendix 2D for Tris·C1)
  • 5% to 10% PhiX (Illumina) or other balanced genome
  • 2 N NaOH
  • Ice‐cold hybridization buffer (HT1, Illumina)
  • 1.5‐ml microcentrifuge tubes
  • 25°C incubator
  • Vortexer
  • Illumina MiSeq platform
  • 500‐µl HandiStepper and tips

Support Protocol 3: Quantification of PCR DNA Using Pico Green

  Materials
  • Stock λ DNA (100 mg/ml)
  • 1× Tris‐EDTA (TE) buffer ( appendix 2D)
  • Sample
  • Pico Green solution (Invitrogen, cat. no. P11495)
  • 96‐tube racks
  • 2‐ml screw‐top tubes with caps
  • 96‐well dilution plates
  • 96‐well, deep‐well plates
  • Multi‐channel pipettor
  • Plate sealer
  • Vortexer
  • Centrifuge and plate rotor
  • Aluminum foil
  • 15‐ml conical tube
  • 50‐ml pipet trough
  • Tecan Infinite M200 fluorescent plate reader or equivalent

Basic Protocol 7: Installing QWRAP and Other Dependencies

Basic Protocol 8: Microbiome Analysis Using QWRAP

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Figures

Videos

Literature Cited

Literature Cited
  Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403‐410.
  Arumugam, M., Raes, J., Pelletier, E., Le Paslier, D., Yamada, T., Mende, D.R., Fernandes, G.R., Tap, J., Bruls, T., Batto, J.M., Bertalan, M., Borruel, N., Casellas, F., Fernandez, L., Gautier, L., Hansen, T., Hattori, M., Hayashi, T., Kleerebezem, M., Kurokawa, K., Leclerc, M., Levenez, F., Manichanh, C., Nielsen, H.B., Nielsen, T., Pons, N., Poulain, J., Qin, J., Sicheritz‐Ponten, T., Tims, S., Torrents, D., Ugarte, E., Zoetendal, E.G., Wang, J., Guarner, F., Pedersen, O., de Vos, W.M., Brunak, S., Dore, J., Antolin, M., Artiguenave, F., Blottiere, H.M., Almeida, M., Brechot, C., Cara, C., Chervaux, C., Cultrone, A., Delorme, C., Denariaz, G., Dervyn, R., Foerstner, K.U., Friss, C., van de Guchte, M., Guedon, E., Haimet, F., Huber, W., van Hylckama‐Vlieg, J., Jamet, A., Juste, C., Kaci, G., Knol, J., Lakhdari, O., Layec, S., Le Roux, K., Maguin, E., Merieux, A., Melo Minardi, R., M'Rini, C., Muller, J., Oozeer, R., Parkhill, J., Renault, P., Rescigno, M., Sanchez, N., Sunagawa, S., Torrejon, A., Turner, K., Vandemeulebrouck, G., Varela, E., Winogradsky, Y., Zeller, G., Weissenbach, J., Ehrlich, S.D., and Bork, P. 2011. Enterotypes of the human gut microbiome. Nature 473:174‐180.
  Bokulich, N.A., Subramanian, S., Faith, J.J., Gevers, D., Gordon, J.I., Knight, R., Mills, D.A., and Caporaso, J.G. 2013. Quality‐filtering vastly improves diversity estimates from Illumina amplicon sequencing. Nat. Methods 10:57‐59.
  Caporaso, J.G., Bittinger, K., Bushman, F.D., DeSantis, T.Z., Andersen, G.L., and Knight, R. 2010a. PyNAST: A flexible tool for aligning sequences to a template alignment. Bioinformatics 26:266‐267.
  Caporaso, J.G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F.D., Costello, E.K., Fierer, N., Pena, A.G., Goodrich, J.K., Gordon, J.I., Huttley, G.A., Kelley, S.T., Knights, D., Koenig, J.E., Ley, R.E., Lozupone, C.A., McDonald, D., Muegge, B.D., Pirrung, M., Reeder, J., Sevinsky, J.R., Turnbaugh, P.J., Walters, W.A., Widmann, J., Yatsunenko, T., Zaneveld, J., and Knight, R. 2010b. QIIME allows analysis of high‐throughput community sequencing data. Nat. Methods 7:335‐336.
  Caporaso, J.G., Lauber, C.L., Walters, W.A., Berg‐Lyons, D., Lozupone, C.A., Turnbaugh, P.J., Fierer, N., and Knight, R. 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.
  Caporaso, J.G., Lauber, C.L., Walters, W.A., Berg‐Lyons, D., Huntley, J., Fierer, N., Owens, S.M., Betley, J., Fraser, L., Bauer, M., Gormley, N., Gilbert, J.A., Smith, G., and Knight, R. 2012. Ultra‐high‐throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J. 6:1621‐1624.
  Cho, I. and Blaser, M.J. 2012. The human microbiome: At the interface of health and disease. Nat. Rev. Genet. 13:260‐270.
  Cole, J.R., Wang, Q., Cardenas, E., Fish, J., Chai, B., Farris, R.J., Kulam‐Syed‐Mohideen, A.S., McGarrell, D.M., Marsh, T., Garrity, G.M., and Tiedje, J.M. 2009. The Ribosomal Database Project: Improved alignments and new tools for rRNA analysis. Nucleic Acids Res. 37:D141‐145.
  Cole, J.R., Wang, Q., Fish, J.A., Chai, B., McGarrell, D.M., Sun, Y., Brown, C.T., Porras‐Alfaro, A., Kuske, C.R., and Tiedje, J.M. 2014. Ribosomal Database Project: Data and tools for high throughput rRNA analysis. Nucleic Acids Res. 41:D633‐D642.
  Coussens, L.M. and Werb, Z. 2002. Inflammation and cancer. Nature 420:860‐867.
  DeSantis, T.Z., Hugenholtz, P., Larsen, N., Rojas, M., Brodie, E.L., Keller, K., Huber, T., Dalevi, D., Hu, P., and Andersen, G.L. 2006. Greengenes, a chimera‐checked 16S rRNA gene database and workbench compatible with ARB. Appl. Environ. Microbiol. 72:5069‐5072.
  Eckburg, P.B., Bik, E.M., Bernstein, C.N., Purdom, E., Dethlefsen, L., Sargent, M., Gill, S.R., Nelson, K.E., and Relman, D.A. 2005. Diversity of the human intestinal microbial flora. Science 308:1635‐1638.
  Edgar, R.C. 2004. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32:1792‐1797.
  Edgar, R.C. 2010. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26:2460‐2461.
  Evans, J., Sheneman, L., and Foster, J. 2006. Relaxed neighbor joining: A fast distance‐based phylogenetic tree construction method. J. Mol. Evol. 62:785‐792.
  Faith, J.J., Guruge, J.L., Charbonneau, M., Subramanian, S., Seedorf, H., Goodman, A.L., Clemente, J.C., Knight, R., Heath, A.C., Leibel, R.L., Rosenbaum, M., and Gordon, J.I. 2013. The long‐term stability of the human gut microbiota. Science 341:1237439.
  Goodman, A.L., Kallstrom, G., Faith, J.J., Reyes, A., Moore, A., Dantas, G., and Gordon, J.I. 2011. Extensive personal human gut microbiota culture collections characterized and manipulated in gnotobiotic mice. Proc. Natl. Acad. Sci. U.S.A. 108:6252‐6257.
  Haas, B.J., Garges, S., Earl, A.M., Feldgarden, M., Ward, D.V., Giannoukos, G., Ciulla, D., Tabbaa, D., Highlander, S.K., Sodergren, E., Methe, B., DeSantis, T.Z., Consotium, H.M., Petrosino, J.F., Knight, R., and Birren, B.W. 2011. Chimeric 16S rRNA sequence formation and detection in Sanger and 454‐pyrosequenced PCR amplicons. Genome Res. 21:494‐504.
  Huse, S.M., Mark Welch, D.B., Voorhis, A., Shipunova, A., Morrison, H.G., Eren, A.M., and Sogin, M.L. 2014. VAMPS: A website for visualization and analysis of microbial population structures. BMC Bioinformatics doi: 10.1186/1471‐2105‐15‐41.
  Huttenhower, C., Gevers, D., Knight, R., Abubucker, S., Badger, J.H., Chinwalla, A.T., Creasy, H.H., Earl, A.M., FitzGerald, M.G., Fulton, R.S., Giglio, M.G., Hallsworth‐Pepin, K., Huttenhower, C., Gevers, D., Knight, R., Abubucker, S., Badger, J.H., Chinwalla, A.T., Creasy, H.H., Earl, A.M., FitzGerald, M.G., Fulton, R.S., Giglio, M.G., Hallsworth‐Pepin, K., Lobos, E.A., Madupu, R., Magrini, F., Martin, J.C., Mitreva, M., Muzny, D.M., Sodergren, E.J., Versalovic, J., Wollam, A.M., Worley, K.C., Wortman, J.R., Young, S.K., Zeng, Q., Aagaard, K.M., Abolude, O.O., Allen‐Vercoe, E., Alm, E.J., Alvarado, L., Andersen, G.L., Anderson, S., Appelbaum, E., Arachchi, H.M., Armitage, G., Arze, C.A., Ayvaz, T., Baker, C.C., Begg, L., Belachew, T., Bhonagiri, V., Bihan, M., Blaser, M.J., Bloom, T., Bonazzi, V., Brooks, J.P., Buck, G.A., Buhay, C.J., Busam, D.A., Campbell, J.L., Canon, S.R., Cantarel, B.L., Chain, P.S.G., Chen, I.A., Chen, L., Chhibba, S., Chu, K., Ciulla, D.M., Clemente, J.C., Clifton, S.W., Conlan, S., Crabtree, J., Cutting, M.A., Davidovics, N.J., Davis, C.C., DeSantis, T.Z., Deal, C., Delehaunty, K.D., Dewhirst, F.E., Deych, E., Ding, Y., Dooling, D.J., Dugan, S.P., Dunne, W.M., Durkin, A.S., Edgar, R.C., Erlich, R.L., Farmer, C.N., Farrell, R.M., Faust, K., Feldgarden, M., Felix, V.M., Fisher, S., Fodor, A.A., Forney, L.J., Foster, L., Di Francesco, V., Friedman, J., Friedrich, D.C., Fronick, C.C., Fulton, L.L., Gao, H., Garcia, N., Giannoukos, G., Giblin, C., Giovanni, M.Y., Goldberg, J.M., Goll, J., Gonzalez, A., Griggs, A., Gujja, S., Kinder Haake, S., Haas, B.J., Hamilton, H.A., Harris, E.L., Hepburn, T.A., Herter, B., Hoffmann, D.E., Holder, M.E., Howarth, C., Huang, K.H., Huse, S.M., Izard, J., Jansson, J.K., Jiang, H., Jordan, C., Joshi, V., Katancik, J.A., Keitel, W.A., Kelley, S.T., Kells, C., King, N.B., Knights, D., Kong, H.H., Koren, O., Koren, S., Kota, K.C., Kovar, C.L., Kyrpides, N.C., La Rosa, P.S., Lee, S.L., Lemon, K.P., Lennon, N., Lewis, C.M., Lewis, L., Ley, R.E., Li, K., Liolios, K., Liu, B., Liu, Y., Lo, C.‐C., Lozupone, C.A., Lunsford, R.D., Madden, T., Mahurkar, A.A., Mannon, P.J., Mardis, E.R., Markowitz, V.M., Mavromatis, K., McCorrison, J.M., McDonald, D., McEwen, J., McGuire, A.L., McInnes, P., Mehta, T., Mihindukulasuriya, K.A., Miller, J.R., Minx, P.J., Newsham, I., Nusbaum, C., O'Laughlin, M., Orvis, J., Pagani, I., Palaniappan, K., Patel, S.M., Pearson, M., Peterson, J., Podar, M., Pohl, C., Pollard, K.S., Pop, M., Priest, M.E., Proctor, L.M., Qin, X., Raes, J., Ravel, J., Reid, J.G., Rho, M., Rhodes, R., Riehle, K.P., Rivera, M.C., Rodriguez‐Mueller, B., Rogers, Y.‐H., Ross, M.C., Russ, C., Sanka, R.K., Sankar, P., Sathirapongsasuti, J.F., Schloss, J.A., Schloss, P.D., Schmidt, T.M., Scholz, M., Schriml, L., Schubert, A.M., Segata, N., Segre, J.A., Shannon, W.D., Sharp, R.R., Sharpton, T.J., Shenoy, N., Sheth, N.U., Simone, G.A., Singh, I., Smillie, C.S., Sobel, J.D., Sommer, D.D., Spicer, P., Sutton, G.G., Sykes, S.M., Tabbaa, D.G., Thiagarajan, M., Tomlinson, C.M., Torralba, M., Treangen, T.J., Truty, R.M., Vishnivetskaya, T.A., Walker, J., Wang, L., Wang, Z., Ward, D.V., Warren, W., Watson, M.A., Wellington, C., Wetterstrand, K.A., White, J.R., Wilczek‐Boney, K., Wu, Y.Q., Wylie, K.M., Wylie, T., Yandava, C., Ye, L., Ye, Y., Yooseph, S., Youmans, B.P., Zhang, L., Zhou, Y., Zhu, Y., Zoloth, L., Zucker, J.D., Birren, B.W., Gibbs, R.A., Highlander, S.K., Methé, B.A., Nelson, K.E., Petrosino, J.F., Weinstock, G.M., Wilson, R.K., and White, O. 2012. Structure, function and diversity of the healthy human microbiome. Nature 486:207‐214.
  Jost, L. 2007. Partitioning diversity into independent alpha and beta components. Ecology 88:2427‐2439.
  Kozich, J.J., Westcott, S.L., Baxter, N.T., Highlander, S.K., and Schloss, P.D. 2013. Development of a dual‐index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Appl. Environ. Microbiol. 79:5112‐5120.
  Kuczynski, J., Stombaugh, J., Walters, W.A., Gonzalez, A., Caporaso, J.G., and Knight, R. 2011. Using QIIME to analyze 16S rRNA gene sequences from microbial communities. Curr. Protoc. Bioinform. 36:10.7.1‐10.7.20.
  Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J., and Higgins, D.G. 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23:2947‐2948.
  Ley, R.E. 2010. Obesity and the human microbiome. Curr. Opin. Gastroenterol. 26:5‐11.
  Li, W. and Godzik, A. 2006. Cd‐hit: A fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 22:1658‐1659.
  Littman, D.R. and Pamer, E.G., 2011. Role of the commensal microbiota in normal and pathogenic host immune responses. Cell Host Microbe 10:311‐323.
  Lozupone, C., Hamady, M., and Knight, R. 2006. UniFrac—An online tool for comparing microbial community diversity in a phylogenetic context. BMC Bioinform. 7:371.
  Lozupone, C.A., Hamady, M., Kelley, S.T., and Knight, R. 2007. Quantitative and qualitative beta diversity measures lead to different insights into factors that structure microbial communities. Appl. Environ. Microbiol. 73:1576‐1585.
  Lozupone, C.A., Stombaugh, J.I., Gordon, J.I., Jansson, J.K., and Knight, R. 2012. Diversity, stability and resilience of the human gut microbiota. Nature 489:220‐230.
  Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H., Yadhukumar, Buchner, A., Lai, T., Steppi, S., Jobb, G., Förster, W., Brettske, I., Gerber, S., Ginhart, A.W., Gross, O., Grumann, S., Hermann, S., Jost, R., König, A., Liss, T., Lüssmann, R., May, M., Nonhoff, B., Reichel, B., Strehlow, R., Stamatakis, A., Stuckmann, N., Vilbig, A., Lenke, M., Ludwig, T., Bode, A., and Schleifer, K.H. 2004. ARB: A software environment for sequence data. Nucleic Acids Res. 32:1363‐1371.
  Magoc, T. and Salzberg, S.L. 2011. FLASH: Fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27:2957‐2963.
  McDonald, D., Price, M.N., Goodrich, J., Nawrocki, E.P., DeSantis, T.Z., Probst, A., Andersen, G.L., Knight, R., and Hugenholtz, P. 2012. An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea. ISME J. 6:610‐618.
  Musso, G., Gambino, R., and Cassader, M. 2011. Interactions between gut microbiota and host metabolism predisposing to obesity and diabetes. Annu. Rev. Med. 62:361‐380.
  Navas‐Molina, J.A., Peralta‐Sanchez, J.M., Gonzalez, A., McMurdie, P.J., Vazquez‐Baeza, Y., Xu, Z., Ursell, L.K., Lauber, C., Zhou, H., Song, S.J., Huntley, J., Ackermann, G.L., Berg‐Lyons, D., Holmes, S., Caporaso, J.G., and Knight, R. 2013. Advancing our understanding of the human microbiome using QIIME. Methods Enzymol. 531:371‐444.
  Nawrocki, E.P., Kolbe, D.L., and Eddy, S.R. 2009. Infernal 1.0: Inference of RNA alignments. Bioinformatics 25:1335‐1337.
  Nechvatal, J.M., Ram, J.L., Basson, M.D., Namprachan, P., Niec, S.R., Badsha, K.Z., Matherly, L.H., Majumdar, A.P., and Kato, I. 2008. Fecal collection, ambient preservation, and DNA extraction for PCR amplification of bacterial and human markers from human feces. J. Microbiol. Methods 72:124‐132.
  Peterson, J., Garges, S., Giovanni, M., McInnes, P., Wang, L., Schloss, J.A., Bonazzi, V., McEwen, J.E., Wetterstrand, K.A., Deal, C., Baker, C.C., Di Francesco, V., Howcroft, T.K., Karp, R.W., Lunsford, R.D., Wellington, C.R., Belachew, T., Wright, M., Giblin, C., David, H., Mills, M., Salomon, R., Mullins, C., Akolkar, B., Begg, L., Davis, C., Grandison, L., Humble, M., Khalsa, J., Little, A.R., Peavy, H., Pontzer, C., Portnoy, M., Sayre, M.H., Starke‐Reed, P., Zakhari, S., Read, J., Watson, B., and Guyer, M. 2009. The NIH Human Microbiome Project. Genome Res. 19:2317‐2323.
  Price, M.N., Dehal, P.S., and Arkin, A.P. 2009. FastTree: Computing large minimum evolution trees with profiles instead of a distance matrix. Mol. Biol. Evol. 26:1641‐1650.
  Schloss, P.D., Westcott, S.L., Ryabin, T., Hall, J.R., Hartmann, M., Hollister, E.B., Lesniewski, R.A., Oakley, B.B., Parks, D.H., Robinson, C.J., Sahl, J.W., Stres, B., Thallinger, G.G., Van Horn, D.J., and Weber, C.F. 2009. Introducing MOTHUR: Open‐source, platform‐independent, community‐supported software for describing and comparing microbial communities. Appl. Environ. Microbiol. 75:7537‐7541.
  Stackebrandt, E. and Goebel, B.M. 1994. Taxonomic note: A place for DNA‐DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int. J. Syst. Bact. 44:846‐849.
  Stamatakis, A., Ludwig, T., and Meier, H. 2005. RAxML‐III: A fast program for maximum likelihood‐based inference of large phylogenetic trees. Bioinformatics 21:456‐463.
  Tilg, H. and Kaser, A. 2011. Gut microbiome, obesity, and metabolic dysfunction. J. Clin. Invest. 121:2126‐2132.
  Tlaskalova‐Hogenova, H., Stepankova, R., Kozakova, H., Hudcovic, T., Vannucci, L., Tuckova, L., Rossmann, P., Hrncir, T., Kverka, M., Zakostelska, Z., Klimesova, K., Pribylova, J., Bartova, J., Sanchez, D., Fundova, P., Borovska, D., Srutkova, D., Zidek, Z., Schwarzer, M., Drastich, P., and Funda, D.P. 2011. The role of gut microbiota (commensal bacteria) and the mucosal barrier in the pathogenesis of inflammatory and autoimmune diseases and cancer: Contribution of germ‐free and gnotobiotic animal models of human diseases. Cell. Mol. Immunol. 8:110‐120.
  Turnbaugh, P.J., Ley, R.E., Mahowald, M.A., Magrini, V., Mardis, E.R., and Gordon, J.I. 2006. An obesity‐associated gut microbiome with increased capacity for energy harvest. Nature 444:1027‐1031.
  Turnbaugh, P.J., Hamady, M., Yatsunenko, T., Cantarel, B.L., Duncan, A., Ley, R.E., Sogin, M.L., Jones, W.J., Roe, B.A., Affourtit, J.P., Egholm, M., Henrissat, B., Heath, A.C., Knight, R., and Gordon, J.I. 2009a. A core gut microbiome in obese and lean twins. Nature 457:480‐484.
  Turnbaugh, P.J., Ridaura, V.K., Faith, J.J., Rey, F.E., Knight, R., and Gordon, J.I. 2009b. The effect of diet on the human gut microbiome: a metagenomic analysis in humanized gnotobiotic mice. Sci. Transl. Med. 1:6ra14.
  Woese, C.R. and Gutell, R.R. 1989. Evidence for several higher order structural elements in ribosomal RNA. Proc. Natl. Acad. Sci. U.S.A. 86:3119‐3122.
  Wu, G.D., Chen, J., Hoffmann, C., Bittinger, K., Chen, Y.Y., Keilbaugh, S.A., Bewtra, M., Knights, D., Walters, W.A., Knight, R., Sinha, R., Gilroy, E., Gupta, K., Baldassano, R., Nessel, L., Li, H., Bushman, F.D., and Lewis, J.D. 2011. Linking long‐term dietary patterns with gut microbial enterotypes. Science 334:105‐108.
Internet Resources
  QIIME: http://www.qiime.org/
  The 16S rRNA gene has been sequenced for a wide range of cultured species and environmental isolates (Woese and Gutell, ; Stackebrandt and Goebel, ). The sequences are publicly available from several rRNA sequence databases including GreenGenes, the Ribosomal Database Project, and Silva (DeSantis et al., ; Cole et al., ; McDonald et al., ) that can be automatically searched for matches to sequences derived from samples of interest.
  GreenGenes: http://greengenes.lbl.gov/cgi‐bin/nph‐index.cgi
  Ribosomal Database Project: http://rdp.cme.msu.edu/
  Silva: http://www.arb‐silva.de/
  UniFrac: http://bmf.colorado.edu/unifrac/
  MOTHUR: http://www.mothur.org/
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