Exploring Microbial Diversity and Taxonomy Using SSU rRNA Hypervariable Tag Sequencing

University of California Davis, United States of America
PLoS Genetics (Impact Factor: 7.53). 12/2008; 4(11):e1000255. DOI: 10.1371/journal.pgen.1000255
Source: PubMed


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.

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    • "The locations sampled are not privately-owned or protected in any way and the field studies did not involve endangered or protected species. In all cases, sequencing data of the V6 region of the bacterial 16S rRNA gene were obtained according to the standardized sequencing pipeline of the ICoMM project (see S2 Table for the primer cocktail used;[35,36]. Fragments were sequenced by pyrosequencing on a Genome Sequencer FLX system (Roche, Basel, Switzerland) at the Marine Biological Laboratory in Woods Hole, MA, USA. Standard flowgram format files (sff) have been deposited in the GenBank Sequence Read Archives ( and their accession numbers are provided in the Supporting Information (S1 Table). "
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    • "Total DNA from sediment and strains was extracted and purified with a FastDNA Spin kit for soil (MP Biomedicals, California, USA) and a DNA purification kit (Tiangen, China), according to the manufacturers' recommendations. The V3 region of bacterial 16S rRNA gene was amplified using primers 338F (5=-ACTCCTACGGGAGGCAGCAG-3=) and 533R (5=- TTACCGCGGCTGCTGGCAC-3=) with identified barcodes (Huse et al. 2008). PCR amplifications were performed using a thermocycler (Eppendorf, Hamburg, "

    Full-text · Dataset · Dec 2015
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    • "UChime analysis against a reference database (ChimeraSlayer GOLD) was used to eliminate chimeras (Edgar et al., 2011). The Global Alignment for Sequence Taxonomy (GAST) assigned taxonomic affiliation (Huse et al., 2008) using SILVA 111 as the reference database (Quast et al., 2013). All V6-V4 data can be accessed via the Visualization and Analysis of Microbial Population Structures website at the MBL in Woods Hole, MA, USA (http://vamps. "
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