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Serial analysis of V6 ribosomal sequence tags (SARST-V6): a method for efficient, high-throughput analysis of microbial community composition

Authors

  • David T. Kysela,

    1. The Josephine Bay Paul Center, Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543, USA.
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    • Present address: Department of Ecology and Evolutionary Biology, Yale University, 165 Prospect Street, New Haven, CT 06511, USA.

  • Carmen Palacios,

    1. The Josephine Bay Paul Center, Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543, USA.
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  • Mitchell L. Sogin

    Corresponding author
    1. The Josephine Bay Paul Center, Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543, USA.
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*E-mail sogin@evol5.mbl.edu; Tel. 508-289-7246; Fax 508-457-4727.

Summary

Serial analysis of ribosomal sequence tags (SARST) is a novel technique for characterizing microbial community composition. The SARST method captures sequence information from concatemers of short 16S rDNA polymerase chain reaction (PCR) amplicons from complex populations of DNA. Here, we describe a similar method, serial analysis of V6 ribosomal sequence tags (SARST-V6), which targets the V6 hypervariable  region  of  bacterial  16S  rRNA  genes. The SARST-V6 technique exploits internal primer sequences to generate compatible restriction digest overhangs, thereby improving upon the efficiency of SARST. Serial analysis of V6 ribosomal sequence tags of bacterial community composition in hydrothermal marine sediments from Guaymas Basin resembled results of cloning and sequencing of single, full-length PCR products from ribosomal RNA genes of the same microbial community. Both methods identified the same major bacterial groups, but only SARST-V6 recovered thermodesulfobacteria and γ-proteobacteria sequences, while only full-length PCR product cloning recovered candidate division OP11 se-quences. There were differences in the relative frequencies of some phylotypes. The disparities reflect differences in the amplicon pool obtained during initial amplification that may result from different primer affinities or DNA degradation. These results demonstrate the utility of SARST-V6 in collecting taxonomically informative data for high-throughput analysis of microbial communities.

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