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UMD CMSC 828G - Microbial community profiling for human microbiome projects

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10.1101/gr.085464.108Access the most recent version at doi: 2009 19: 1141-1152 originally published online April 21, 2009Genome Res. Micah Hamady and Rob Knight Tools, techniques, and challengesMicrobial community profiling for human microbiome projects: References http://genome.cshlp.org/content/19/7/1141.full.html#ref-list-1This article cites 109 articles, 59 of which can be accessed free at:serviceEmail alerting click heretop right corner of the article orReceive free email alerts when new articles cite this article - sign up in the box at the http://genome.cshlp.org/subscriptions go to: Genome ResearchTo subscribe to Copyright © 2009 by Cold Spring Harbor Laboratory Press Cold Spring Harbor Laboratory Press on August 14, 2009 - Published by genome.cshlp.orgDownloaded fromNext-Generation DNA Sequencing/ReviewMicrobial community profiling for humanmicrobiome projects: Tools, techniques, and challengesMicah Hamady1and Rob Knight2,31Department of Computer Science, University of Colorado, Boulder, Colorado 80309, USA;2Department of Chemistryand Biochemistry, University of Colorado, Boulder, Colorado 80309, USAHigh-throughput sequencing studies and new software tools are revolutionizing microbial community analyses, yet thevariety of experimental and computational methods can be daunting. In this review, we discuss some of the differentapproaches to community profiling, highlighting strengths and weaknesses of various experimental approaches, se-quencing methodologies, and analytical methods. We also address one key question emerging from various HumanMicrobiome Projects: Is there a substantial core of abundant organisms or lineages that we all share? It appears that insome human body habitats, such as the hand and the gut, the diversity among individuals is so great that we can rule outthe possibility that any species is at high abundance in all individuals: It is possible that the focus should instead be onhigher-level taxa or on functional genes instead.The human microbiota (the collection of microbes that live onand inside us) consists of about 100 trillion microbial cells thatoutnumber our ‘‘human’’ cells 10 to 1 (Savage 1977), and thatprovide a wide range of metabolic functions that we lack (Gill et al.2006). If we consider ourselves as supraorganisms encompassingthese microbial symbionts (Lederberg 2000), by far the majority ofgenes in the system are microbial. In this sense, completing thehuman genome requires us to characterize the microbiome (thecollection of genes in the microbiota) (Turnbaugh et al. 2007).Currently, there are two main methods for performing this char-acterization that do not rely on growing organisms in pure culture:small-subunit ribosomal RNA (rRNA) studies, in which the 16SrRNA gene sequences (for archaea and bacteria) or the 18S rRNAgene sequences (for eukaryotes) are used as stable phylogeneticmarkers to define which lineages are present in a sample (Pace1997), and metagenomic studies, in which community DNA issubject to shotgun sequencing (Rondon et al. 2000). Small sub-unit rRNA-based studies are sometimes also considered to be‘‘metagenomic’’ in that they analyze a heterogeneous sample ofcommunity DNA. Community profiling, or determining theabundance of each kind of microbe, is much cheaper usingrRNA because only one gene out of each genome is examined,but metagenomic profiles are essential for understanding thefunctions encoded in those genomes. Techniques that probegene expression directly such as metatranscriptomics and meta-proteomics (analysis of the transcripts or proteins in a community,respectively), although useful in simpler microbial communitiessuch as acid mine drainage (Lo et al. 2007; Frias-Lopez et al. 2008),are just beginning to be applied to human-associated microbialcommunities (Verberkmoes et al. 2008).Through the use of metagenomic and rRNA-based techni-ques, much progress has been made in characterizing the humanmicrobiome and its role in health and disease in the past few years,especially with the advent of high-throughput sequencing. Thesestudies are challenging because of the scale and complexity of themicrobiome and because of the unexpected variability betweenindividuals. In this review, we cover the combination of experi-mental and analytical techniques used to characterize the micro-biomes of humans and of other mammals. In particular, wedescribe how recent advances in technology and experimentaltechniques, together with computational methods that draw onthe long tradition of community analysis in large-scale ecologicalstudies, are essential for uncovering large-scale trends that relatethe microbiomes of many individuals.One fundamental question raised by the National Institutesof Health (NIH), European Union (EU), and other sponsored Hu-man Microbiome Projects (HMPs) is whether there is a core hu-man microbiome of genes or species that we all share (Fig. 1;Turnbaugh et al. 2007, 2009). If there is a substantial core, thestrategy for understanding the microbiome is clear: Identify theorganisms that comprise the core using 16S rRNA analysis, se-quence their genomes, and use these genomes as scaffolds formetagenomic, metatranscriptomic, and metaproteomic studiesthat provide information about small fragments of genes, tran-scripts, or proteins, respectively, but that require assembly againstknown sequences (Turnbaugh et al. 2007; Zaneveld et al. 2008).However, if there is a minimal core or no core at all, alternativestrategies will need to be developed because new genes and specieswill continue to be found in each new person examined.Another key question is whether changes in the relativeabundance of members of human-associated microbial commu-nities are generally important. For example, the proportionalrepresentation of the bacterial phyla Firmicutes, Actinobacteria,and Bacteroidetes in the gut is associated with obesity in bothhumans and mice (Ley et al. 2005, 2006c; Turnbaugh et al.2006, 2008). However, although this observation establishes thatchanges in the abundance of broad bacterial groups such as entirephyla can be important and we also know that miniscule inocu-lations of particular pathogenic strains can cause disease, we knowlittle about the physiological impacts of changes in microbialabundance at a given taxonomic level in general. Our power todetect particular species depends on depth of sequencing and onwhether they can be selected by culture-based techniques. Forexample, we think of


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UMD CMSC 828G - Microbial community profiling for human microbiome projects

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