Exploring Microbial Diversity and Taxonomy Using SSU rRNA Hypervariable Tag Sequencing

Susan M Huse,Les Dethlefsen,Mitchell L Sogin,David Mark Welch,Julie A Huber,David A Relman,Jonathan A Eisen

doi:10.1371/journal.pgen.1000255

Abstract

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.

Highlights

The biosphere contains between 1030 and 1031 microbial genomes, at least 2–3 orders of magnitude more than the number of plant and animal cells combined [1]
In recognition of the role marine microbes play in the biogeochemical processes that are critical to life in all environments on Earth including carbon and nitrogen cycling, the International Census of Marine Microbes (ICoMM: http://icomm.mbl.edu) has launched an international effort to catalogue the diversity of microbial populations in the oceanic, coastal, and benthic waters
Phylogenetic trees based on 16S ribosomal RNA genes have been used with great success to identify microbial taxonomy from DNA alone

Summary

Introduction

The biosphere contains between 1030 and 1031 microbial genomes, at least 2–3 orders of magnitude more than the number of plant and animal cells combined [1]. Microbes control global utilization of nitrogen through nitrogen fixation, nitrification, and nitrate reduction, and drive the bulk of sulfur, iron and manganese biogeochemical cycles [2]. They regulate the composition of the atmosphere, influence climates, recycle nutrients, and decompose pollutants. In recognition of the role marine microbes play in the biogeochemical processes that are critical to life in all environments on Earth including carbon and nitrogen cycling, the International Census of Marine Microbes (ICoMM: http://icomm.mbl.edu) has launched an international effort to catalogue the diversity of microbial populations in the oceanic, coastal, and benthic waters. Other recent human health studies include microbes in breast milk [18], chronic wounds [19], human gut [20], dental caries [21], and childcare facilities [22]

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Discussion

Conclusion