Exploring Microbial Diversity and Taxonomy Using SSU rRNA Hypervariable Tag Sequencing

Massively parallel pyrosequencing of hypervariable regions from small subunit ribosomal RNA (SSU rRNA) genes can sample a microbial community two or three orders of magnitude more deeply per dollar and per hour than capillary sequencing of full-length SSU rRNA. As with full-length rRNA surveys, each...

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Vydáno v:	PLoS genetics Ročník 4; číslo 11; s. e1000255
Hlavní autoři:	Huse, Susan M., Dethlefsen, Les, Huber, Julie A., Welch, David Mark, Relman, David A., Sogin, Mitchell L.
Médium:	Journal Article
Jazyk:	angličtina
Vydáno:	United States Public Library of Science 01.11.2008 Public Library of Science (PLoS)
Témata:	Bacteria - classification Bacteria - genetics Biodiversity Biosphere Biotechnology/Applied Microbiology Biotechnology/Environmental Microbiology Classification - methods Computational Biology/Comparative Sequence Analysis Computational Biology/Metagenomics Computational Biology/Molecular Genetics Deoxyribonucleic acid DNA DNA sequencing Ecology/Community Ecology and Biodiversity Ecology/Environmental Microbiology Evolutionary Biology/Bioinformatics Evolutionary Biology/Microbial Evolution and Genomics Experiments Genes Genetics and Genomics/Microbial Evolution and Genomics Humans Metagenome - genetics Methods Microbial genetics Microbiology Microbiology/Applied Microbiology Microbiology/Environmental Microbiology Microbiology/Medical Microbiology Microbiology/Microbial Evolution and Genomics Nitrogen Nucleotide sequencing Physiological aspects Ribosomal RNA RNA, Ribosomal - genetics Sequence Analysis, DNA Sequence Tagged Sites Taxonomy United States
ISSN:	1553-7404, 1553-7390, 1553-7404
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Popis
Shrnutí:	Massively parallel pyrosequencing of hypervariable regions from small subunit ribosomal RNA (SSU rRNA) genes can sample a microbial community two or three orders of magnitude more deeply per dollar and per hour than capillary sequencing of full-length SSU rRNA. As with full-length rRNA surveys, each sequence read is a tag surrogate for a single microbe. However, rather than assigning taxonomy by creating gene trees de novo that include all experimental sequences and certain reference taxa, we compare the hypervariable region tags to an extensive database of rRNA sequences and assign taxonomy based on the best match in a Global Alignment for Sequence Taxonomy (GAST) process. The resulting taxonomic census provides information on both composition and diversity of the microbial community. To determine the effectiveness of using only hypervariable region tags for assessing microbial community membership, we compared the taxonomy assigned to the V3 and V6 hypervariable regions with the taxonomy assigned to full-length SSU rRNA sequences isolated from both the human gut and a deep-sea hydrothermal vent. The hypervariable region tags and full-length rRNA sequences provided equivalent taxonomy and measures of relative abundance of microbial communities, even for tags up to 15% divergent from their nearest reference match. The greater sampling depth per dollar afforded by massively parallel pyrosequencing reveals many more members of the "rare biosphere" than does capillary sequencing of the full-length gene. In addition, tag sequencing eliminates cloning bias and the sequences are short enough to be completely sequenced in a single read, maximizing the number of organisms sampled in a run while minimizing chimera formation. This technique allows the cost-effective exploration of changes in microbial community structure, including the rare biosphere, over space and time and can be applied immediately to initiatives, such as the Human Microbiome Project.
Bibliografie:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 ObjectType-Article-2 ObjectType-Feature-1 Conceived and designed the experiments: SMH DMW DAR MLS. Performed the experiments: SMH LD JAH. Analyzed the data: SMH DMW. Contributed reagents/materials/analysis tools: SMH. Wrote the paper: SMH DMW DAR MLS.
ISSN:	1553-7404 1553-7390 1553-7404
DOI:	10.1371/journal.pgen.1000255