Microbiome 101: Studying, Analyzing, and Interpreting Gut Microbiome Data for Clinicians

&NA; Advances in technical capabilities for reading complex human microbiomes are leading to an explosion of microbiome research, leading in turn to intense interest among clinicians in applying these techniques to their patients. In this review, we discuss the content of the human microbiome, including intersubject and intrasubject variability, considerations of study design including important confounding factors, and different methods in the laboratory and on the computer to read the microbiome and its resulting gene products and metabolites. We highlight several common pitfalls for clinicians, including the expectation that an individual’s microbiome will be stable, that diet can induce rapid changes that are large compared with the differences among subjects, that everyone has essentially the same core stool microbiome, and that different laboratory and computational methods will yield essentially the same results. We also highlight the current limitations and future promise of these techniques, with the expectation that an understanding of these considerations will help accelerate the path toward routine clinical application of these techniques developed in research settings.

[1]  Rob Knight,et al.  Selection of primers for optimal taxonomic classification of environmental 16S rRNA gene sequences , 2012, The ISME Journal.

[2]  Rob Knight,et al.  Bayesian community-wide culture-independent microbial source tracking , 2011, Nature Methods.

[3]  R. Knight,et al.  Accurate taxonomy assignments from 16S rRNA sequences produced by highly parallel pyrosequencers , 2008, Nucleic acids research.

[4]  F. Bushman,et al.  Linking Long-Term Dietary Patterns with Gut Microbial Enterotypes , 2011, Science.

[5]  C. Huttenhower,et al.  Assessment of variation in microbial community amplicon sequencing by the Microbiome Quality Control (MBQC) project consortium , 2017, Nature Biotechnology.

[6]  J Stypułkowski,et al.  Profile of Gut Microbiota Associated With the Presence of Hepatocellular Cancer in Patients With Liver Cirrhosis. , 2016, Transplantation proceedings.

[7]  P. Turnbaugh,et al.  Microbial ecology: Human gut microbes associated with obesity , 2006, Nature.

[8]  R. Cichewicz,et al.  Fungal biofilm inhibitors from a human oral microbiome-derived bacterium. , 2012, Organic & biomolecular chemistry.

[9]  C. Huttenhower,et al.  PhyloPhlAn is a new method for improved phylogenetic and taxonomic placement of microbes , 2013, Nature Communications.

[10]  E. Purdom,et al.  Diversity of the Human Intestinal Microbial Flora , 2005, Science.

[11]  Mathieu Almeida,et al.  Dietary intervention impact on gut microbial gene richness , 2013, Nature.

[12]  Curtis Huttenhower,et al.  bioBakery: a meta’omic analysis environment , 2017, Bioinform..

[13]  B. Leggett,et al.  Numerical ecology validates a biogeographical distribution and gender-based effect on mucosa-associated bacteria along the human colon , 2011, The ISME Journal.

[14]  B. Roe,et al.  A core gut microbiome in obese and lean twins , 2008, Nature.

[15]  R. Knight,et al.  The Human Microbiome Project , 2007, Nature.

[16]  Jianxin Shi,et al.  Collecting Fecal Samples for Microbiome Analyses in Epidemiology Studies , 2015, Cancer Epidemiology, Biomarkers & Prevention.

[17]  Lawrence A. David,et al.  Diet rapidly and reproducibly alters the human gut microbiome , 2013, Nature.

[18]  James T. Morton,et al.  Microbiome-wide association studies link dynamic microbial consortia to disease , 2016, Nature.

[19]  Patrick D. Schloss,et al.  Looking for a Signal in the Noise: Revisiting Obesity and the Microbiome , 2016, mBio.

[20]  Z. Ma,et al.  Spatial heterogeneity and co-occurrence patterns of human mucosal-associated intestinal microbiota , 2013, The ISME Journal.

[21]  Kristian Fog Nielsen,et al.  Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking , 2016, Nature Biotechnology.

[22]  J. Handelsman,et al.  Metagenomics: genomic analysis of microbial communities. , 2004, Annual review of genetics.

[23]  Rob Knight,et al.  Comparison of Collection Methods for Fecal Samples for Discovery Metabolomics in Epidemiologic Studies , 2016, Cancer Epidemiology, Biomarkers & Prevention.

[24]  Nuno Bandeira,et al.  Mass spectral molecular networking of living microbial colonies , 2012, Proceedings of the National Academy of Sciences.

[25]  R. Knight,et al.  Global chemical analysis of biology by mass spectrometry , 2017 .

[26]  Lisa M Bramer,et al.  Dynamics of the human gut microbiome in Inflammatory Bowel Disease , 2017, Nature Microbiology.

[27]  R. Knight,et al.  Moving pictures of the human microbiome , 2011, Genome Biology.

[28]  Wei Jia,et al.  Distinctly altered gut microbiota in the progression of liver disease , 2016, Oncotarget.

[29]  Rosaleen J. Anderson,et al.  Methods for the detection and identification of pathogenic bacteria: past, present, and future. , 2017, Chemical Society reviews.

[30]  Karsten Zengler,et al.  Improving saliva shotgun metagenomics by chemical host DNA depletion , 2018, Microbiome.

[31]  W. Ludwig,et al.  SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB , 2007, Nucleic acids research.

[32]  Luke R. Thompson,et al.  Best practices for analysing microbiomes , 2018, Nature Reviews Microbiology.

[33]  Justine W. Debelius,et al.  The gut–liver axis and the intersection with the microbiome , 2018, Nature Reviews Gastroenterology & Hepatology.

[34]  Kunihiko Sadakane,et al.  MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph , 2014, Bioinform..

[35]  Daniel H. Huson,et al.  MEGAN Community Edition - Interactive Exploration and Analysis of Large-Scale Microbiome Sequencing Data , 2016, PLoS Comput. Biol..

[36]  Robin Sen,et al.  UNITE: a database providing web-based methods for the molecular identification of ectomycorrhizal fungi. , 2005, The New phytologist.

[37]  William A. Walters,et al.  Conducting a Microbiome Study , 2014, Cell.

[38]  P. Bork,et al.  Enterotypes of the human gut microbiome , 2011, Nature.

[39]  Jun Wang,et al.  Disentangling type 2 diabetes and metformin treatment signatures in the human gut microbiota , 2015, Nature.

[40]  J. Tiedje,et al.  Naïve Bayesian Classifier for Rapid Assignment of rRNA Sequences into the New Bacterial Taxonomy , 2007, Applied and Environmental Microbiology.

[41]  Rob Knight,et al.  Guiding longitudinal sampling in IBD cohorts , 2017, Gut.

[42]  S. Salzberg,et al.  Centrifuge: rapid and sensitive classification of metagenomic sequences , 2016, bioRxiv.

[43]  James R. Cole,et al.  Ribosomal Database Project: data and tools for high throughput rRNA analysis , 2013, Nucleic Acids Res..

[44]  J. Nicholson,et al.  Sex-dependent effects on gut microbiota regulate hepatic carcinogenic outcomes , 2017, Scientific Reports.

[45]  Katherine H. Huang,et al.  Structure, Function and Diversity of the Healthy Human Microbiome , 2012, Nature.

[46]  J. Clemente,et al.  Gut Microbiota from Twins Discordant for Obesity Modulate Metabolism in Mice , 2013, Science.

[47]  Rohit Loomba,et al.  Inflammation-induced IgA+ cells dismantle anti-liver cancer immunity , 2017, Nature.

[48]  Timothy J. Laurent,et al.  A Taxonomic Signature of Obesity in the Microbiome? Getting to the Guts of the Matter , 2014, PloS one.

[49]  Jianxin Shi,et al.  Comparison of Collection Methods for Fecal Samples in Microbiome Studies , 2017, American journal of epidemiology.

[50]  R. Knight,et al.  Dog and human inflammatory bowel disease rely on overlapping yet distinct dysbiosis networks , 2016, Nature Microbiology.

[51]  Susan P. Holmes,et al.  phyloseq: A Bioconductor Package for Handling and Analysis of High-Throughput Phylogenetic Sequence Data , 2011, Pacific Symposium on Biocomputing.

[52]  Bernard Henrissat,et al.  Metabolic Reconstruction for Metagenomic Data and Its Application to the Human Microbiome , 2012, PLoS Comput. Biol..

[53]  K. Zengler,et al.  Cultivating the uncultured , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[54]  M. Bassetti,et al.  New antibiotics for bad bugs: where are we? , 2013, Annals of Clinical Microbiology and Antimicrobials.

[55]  William A. Walters,et al.  Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms , 2012, The ISME Journal.

[56]  Rob Knight,et al.  Regional variation limits applications of healthy gut microbiome reference ranges and disease models , 2018, Nature Medicine.

[57]  D. Harris,et al.  Rapid identification of bacteria and candida using pna-fish from blood and peritoneal fluid cultures: a retrospective clinical study , 2013, Annals of Clinical Microbiology and Antimicrobials.

[58]  D. Savage Microbial ecology of the gastrointestinal tract. , 1977, Annual review of microbiology.

[59]  Richard A. Flavell,et al.  Inflammasome-mediated dysbiosis regulates progression of NAFLD and obesity , 2012, Nature.

[60]  Rick L. Stevens,et al.  A communal catalogue reveals Earth’s multiscale microbial diversity , 2017, Nature.

[61]  R. Knight,et al.  Meta‐analyses of human gut microbes associated with obesity and IBD , 2014, FEBS letters.

[62]  Timothy L. Tickle,et al.  Computational meta'omics for microbial community studies , 2013, Molecular systems biology.

[63]  P. Gentileschi,et al.  Molecular Phenomics and Metagenomics of Hepatic Steatosis in Non-Diabetic Obese Women , 2018, Nature Medicine.

[64]  William A. Walters,et al.  QIIME allows analysis of high-throughput community sequencing data , 2010, Nature Methods.

[65]  Robin H. Liu,et al.  Self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and DNA microarray detection. , 2004, Analytical chemistry.

[66]  C. Nakatsu,et al.  Microbial Ecology along the Gastrointestinal Tract , 2017, Microbes and environments.

[67]  N. Pace,et al.  Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases , 2007, Proceedings of the National Academy of Sciences.

[68]  R. Knight,et al.  Tracking Human Gut Microbiome Changes Resulting from a Colonoscopy , 2017, Methods of Information in Medicine.

[69]  Jose A Navas-Molina,et al.  Deblur Rapidly Resolves Single-Nucleotide Community Sequence Patterns , 2017, mSystems.

[70]  P. Bork,et al.  A human gut microbial gene catalogue established by metagenomic sequencing , 2010, Nature.

[71]  Sergey I. Nikolenko,et al.  SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing , 2012, J. Comput. Biol..

[72]  Nitin Kumar,et al.  Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation , 2016, Nature.

[73]  H. Frickmann,et al.  More Pathogenicity or Just More Pathogens?—On the Interpretation Problem of Multiple Pathogen Detections with Diagnostic Multiplex Assays , 2017, Front. Microbiol..

[74]  Derrick E. Wood,et al.  Kraken: ultrafast metagenomic sequence classification using exact alignments , 2014, Genome Biology.

[75]  Se Jin Song,et al.  Tiny microbes, enormous impacts: what matters in gut microbiome studies? , 2016, Genome Biology.

[76]  J. Kuhn,et al.  Synthesis and degradation of lac mRNA in E. coli depleted of 30S ribosomal subunits , 1979, Molecular and General Genetics MGG.

[77]  Ryan Hendrickson,et al.  KatharoSeq Enables High-Throughput Microbiome Analysis from Low-Biomass Samples , 2018, mSystems.

[78]  K. Trebesius,et al.  Fluorescent In Situ Hybridization Allows Rapid Identification of Microorganisms in Blood Cultures , 2000, Journal of Clinical Microbiology.

[79]  D Raoult,et al.  Microbial culturomics: paradigm shift in the human gut microbiome study. , 2012, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[80]  R. Milo,et al.  Revised Estimates for the Number of Human and Bacteria Cells in the Body , 2016, bioRxiv.

[81]  Rohit Loomba,et al.  Link between gut‐microbiome derived metabolite and shared gene‐effects with hepatic steatosis and fibrosis in NAFLD , 2018, Hepatology.

[82]  R. Knight,et al.  Global patterns in bacterial diversity , 2007, Proceedings of the National Academy of Sciences.

[83]  Eric A. Franzosa,et al.  Linking the Human Gut Microbiome to Inflammatory Cytokine Production Capacity , 2016, Cell.

[84]  P. Bork,et al.  Richness of human gut microbiome correlates with metabolic markers , 2013, Nature.

[85]  Se Jin Song,et al.  The treatment-naive microbiome in new-onset Crohn's disease. , 2014, Cell host & microbe.

[86]  H. Nishioka,et al.  Radiographic localization of a spontaneous cerebrospinal fluid fistula. Case report. , 1966, Journal of neurosurgery.

[87]  Shibu Yooseph,et al.  Gut Microbiome-Based Metagenomic Signature for Non-invasive Detection of Advanced Fibrosis in Human Nonalcoholic Fatty Liver Disease. , 2017, Cell metabolism.

[88]  Rob Knight,et al.  American Gut: an Open Platform for Citizen Science Microbiome Research , 2018, mSystems.

[89]  Eoin L. Brodie,et al.  Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB , 2006, Applied and Environmental Microbiology.

[90]  Justine W. Debelius,et al.  The Microbiome and Human Biology. , 2017, Annual review of genomics and human genetics.

[91]  R. Knight,et al.  Bacterial Community Variation in Human Body Habitats Across Space and Time , 2009, Science.

[92]  Ivan V. Protsyuk,et al.  Coupling Targeted and Untargeted Mass Spectrometry for Metabolome-Microbiome-Wide Association Studies of Human Fecal Samples. , 2017, Analytical chemistry.

[93]  Jeroen Raes,et al.  Stool consistency is strongly associated with gut microbiota richness and composition, enterotypes and bacterial growth rates , 2015, Gut.

[94]  Tom O. Delmont,et al.  Anvi’o: an advanced analysis and visualization platform for ‘omics data , 2015, PeerJ.

[95]  J. Petrosino,et al.  Microbiota Modulate Behavioral and Physiological Abnormalities Associated with Neurodevelopmental Disorders , 2013, Cell.

[96]  Angela C. Poole,et al.  Human Genetics Shape the Gut Microbiome , 2014, Cell.

[97]  Amnon Amir,et al.  Gut Microbiota Offers Universal Biomarkers across Ethnicity in Inflammatory Bowel Disease Diagnosis and Infliximab Response Prediction , 2018, mSystems.

[98]  Rob Knight,et al.  Longitudinal analysis of microbial interaction between humans and the indoor environment , 2014, Science.

[99]  R. S. Huang,et al.  Performance of the Verigene® enteric pathogens test, Biofire FilmArray™ gastrointestinal panel and Luminex xTAG® gastrointestinal pathogen panel for detection of common enteric pathogens. , 2016, Diagnostic microbiology and infectious disease.

[100]  Tetsuya Hayashi,et al.  Extensive genomic diversity and selective conservation of virulence-determinants in enterohemorrhagic Escherichia coli strains of O157 and non-O157 serotypes , 2007, Genome Biology.

[101]  Eric P. Nawrocki,et al.  An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea , 2011, The ISME Journal.

[102]  Philip Hugenholtz,et al.  A renaissance for the pioneering 16S rRNA gene. , 2008, Current opinion in microbiology.

[103]  Emily S. Charlson,et al.  Minimum information about a marker gene sequence (MIMARKS) and minimum information about any (x) sequence (MIxS) specifications , 2011, Nature Biotechnology.