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Metagenomic Pyrosequencing and Microbial Identification Reviews Clinical Chemistry 55:5 856–866 (2009) Reviews Metagenomic Pyrosequencing and Microbial Identification Joseph F. Petrosino,1,2 Sarah Highlander,1,2 Ruth Ann Luna,3,4 Richard A. Gibbs,2,5 and James Versalovic1,3,4,5,6* BACKGROUND: The Human Microbiome Project has ush- niches, in plants, or in animal hosts. Examples in mam- ered in a new era for human metagenomics and high- malian biology include studies of microbial communi- throughput next-generation sequencing strategies. ties on various mucosal surfaces and on the human skin. This review focuses on analytical strategies for CONTENT: This review describes evolving strategies in identifying pathogens in mixed microbial communi- metagenomics, with a special emphasis on the core ties via metagenomics. technology of DNA pyrosequencing. The challenges of Metagenomics and associated metastrategies have microbial identification in the context of microbial arrived at the forefront of biology primarily because of populations are discussed. The development of next- 2 major developments. The deployment of next- generation pyrosequencing strategies and the technical generation DNA-sequencing technologies in many hurdles confronting these methodologies are addressed. centers has greatly enhanced capabilities for sequenc- Bioinformatics-related topics include taxonomic systems, ing large meta–data sets. Technologic advances have sequence databases, sequence-alignment tools, and clas- created new opportunities for the pursuit of large-scale sifiers. DNA sequencing based on 16S rRNA genes or en- sequencing projects that were difficult to imagine just tire genomes is summarized with respect to potential py- several years ago. The second key development is an rosequencing applications. emerging appreciation for the importance of complex microbial communities in mammalian biology and in SUMMARY: Both the approach of 16S rDNA amplicon human health and disease. The Human Microbiome sequencing and the whole-genome sequencing ap- Project was approved in May 2007 as one of 2 major proach may be useful for human metagenomics, and numerous bioinformatics tools are being deployed to components (in addition to the human epigenomics tackle such vast amounts of microbiological sequence program) of RoadMap version 1.5 of the US NIH (1). diversity. Metagenomics, or genetic studies of micro- The demands of this project have produced intense in- bial communities, may ultimately contribute to a more terest and a focus in genome centers to apply parallel comprehensive understanding of human health, dis- DNA-sequencing technologies to human biology on a ease susceptibilities, and the pathophysiology of infec- scale not previously witnessed. tious and immune-mediated diseases. The human microbiome is the entire population © 2009 American Association for Clinical Chemistry of microbes that colonize the human body, including the gastrointestinal tract, the genitourinary tract, the oral cavity, the nasopharynx, the respiratory tract, and the skin. The different microorganisms constituting Metagenomics and the Human Microbiome the microbiome include bacteria, fungi (mostly yeasts), and viruses. Depending on the context, parasites may Metagenomics refers to culture-independent studies of also be considered to compose part of the indigenous the collective set of genomes of mixed microbial com- microbiota. The “metagenome” of microbial commu- munities and applies to explorations of all microbial nities that occupy various sites in the body is estimated genomes in consortia that reside in environmental to be approximately 100-fold greater in terms of gene content than the human genome. These diverse and complex collections of genes encode a wide array of 1 Department of Molecular Virology and Microbiology, Baylor College of Medi- biochemical and physiological functions that may ben- cine, Houston, TX; 2 Human Genome Sequencing Center, Baylor College of efit the host, as well as neighboring microbes (1).We Medicine, Houston, TX; 3 Department of Pathology, Baylor College of Medicine, Houston, TX; 4 Department of Pathology, Texas Children’s Hospital, Houston, focus on bacterial populations because bacteria form a TX; 5 Department of Molecular and Human Genetics, Baylor College of Medi- predominant group of the microbiome and have the cine, Houston, TX; 6 Department of Pediatrics, Baylor College of Medicine, Houston, TX. most comprehensively documented phylogenetic data * Address correspondence to this author at: Department of Pathology, Texas sets and classification systems. Most of the data gath- Children’s Hospital, 6621 Fannin MC 1-2261, Houston, TX 77030. Fax 832- ered to date have been compiled with Sanger (dideoxy) 825-0164; e-mail [email protected]. Received July 31, 2008; accepted January 28, 2009. sequencing platforms, but this review focuses on Previously published online at DOI: 10.1373/clinchem.2008.107565 emerging parallel DNA-sequencing technologies based 856 Metagenomic Pyrosequencing Reviews on pyrosequencing. Such next-generation sequencing mucosa-associated populations and fecal bacterial systems introduce possibilities for deeply sequenced populations in humans and nonhuman primates (6– data collections and strategies aimed at microbial iden- 12). In addition to differences with respect to sample tification via single genetic targets or whole-genome type or body location, differences have also been noted methodologies. with respect to sex in nonhuman primates, implying Several important issues have recently emerged sexual dimorphism with respect to the human micro- with respect to metagenomics and microbes. One issue biome (12). To address the central challenge of micro- is that the science of metagenomics, in contrast to in- bial identification in the context of mixed species com- dividual microbial or animal genomes, is ultracomplex munities requires refining the primary strategies for and challenged by the existence of vast unknown or DNA sequencing–based bacterial identification. knowledge “deserts.” Of the immense microbial taxo- nomic “space” in nature, only a restricted set of bacte- DNA Sequencing and Bacterial Identification rial populations have been identified in the human body. As an example, the colonic microbiota is a vast Pathogen identification in infectious diseases relies ecosystem with approximately 800–1000 species per mostly on routine cultures and biochemical testing by individual, but these estimates are in flux because the means of semiautomated platforms in the clinical lab- science of metagenomics and microbial pan-arrays is oratory. The shift toward widespread adoption of nu- so new. Approximately 62% of the bacteria identified cleic acid sequencing for identification of microbial from the human intestine were previously unknown, pathogens has been slowed by the user-intensive, and 80% of the bacteria identified by metagenomic se- highly technical nature of Sanger DNA sequencing. quencing were considered noncultivatable (2). Only 9 Nevertheless, several studies published in the 1990s in- of 70 bacterial phyla (divisions that vary in number dicated that sequencing of 16S rRNA genes could be depending on the taxonomic scheme) have been found useful for pathogen discovery and identification in the human intestine, and 2 bacterial phyla, the Fir- (13, 14). Prior studies of bacterial evolution and phy- micutes and the Bacteroidetes, predominate in numbers logenetics provided the foundation for subsequent ap- (1, 3). As an example within the Firmicutes, the genus plications of sequencing based on 16S rRNA genes (or Lactobacillus includes Ͼ100 different species (http:// 16S rDNA) for microbial identification (15). The ini- www.bacterio.cict.fr/l/lactobacillus.html). To date, tial studies were based on Sanger-sequencing strategies fewer than 20 Lactobacillus species have been found that included targeted sequencing of 16S rRNA genes consistently in the mammalian gastrointestinal tract. (approximately 1.5 kb of target sequence). Such “long These findings indicate that membership in indigenous read” approaches enabled investigators and medical communities is restricted to a limited subset of all bac- laboratory scientists to identify many individual genera teria, and bacterial populations are not randomly dis- and species that could not be identified with biochem- tributed in and on the human body. Preliminary stud- ical methods. Sequence-based identification could be ies suggest that the predominant species in the established with a reasonable amount of confidence genitourinary tract and on skin sites are fundamentally from relatively long reads and with the aid of sequence- different from the populations predominant in the gas- classifier algorithms that included most of the 16S trointestinal tract (4, 5). rDNA coding sequence; however, less than half of the In contrast to the human genome, the human met- coding sequence (approximately 500 bp), including agenomes may differ, depending on location (site) several hypervariable regions, may be sufficient for within the human body, age, and such environmental genus- and species-level pathogen identification via factors as diet. A key remaining question is whether a Sanger sequencing (14, 16). As sequence targets for core human microbiome is definable (1). Different re- microbial identification have become more precisely gions of the body, even in related and contiguous sites, defined, the introduction of pyrosequencing has pro- may differ with respect to bacterial quantities and spe- vided a user-friendly approach for the clinical labora-
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