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Applied and Environmental Microbiology, May 2009, p. 2889-2898, Vol. 75, No. 9
0099-2240/09/$08.00+0 doi:10.1128/AEM.01640-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Pyrosequencing of the Chaperonin-60 Universal Target as a Tool for Determining Microbial Community Composition 
,
John Schellenberg,1
Matthew G. Links,2
Janet E. Hill,3
Tim J. Dumonceaux,4*
Geoffrey A. Peters,4
Shaun Tyler,4
T. Blake Ball,1
Alberto Severini,1,4 and
Francis A. Plummer1,4
Department of Medical Microbiology, University of Manitoba, 730 William Avenue, Winnipeg, Manitoba R3E 0W3, Canada,1 Agriculture and Agri-Food Canada Saskatoon Research Centre, 107 Science Place, Saskatoon, Saskatchewan S7N 0X2, Canada,2 Department of Veterinary Microbiology, University of Saskatchewan, 52 Campus Drive, Saskatoon, Saskatchewan S7N 5B4, Canada,3 National Microbiology Laboratory, Canadian Science Centre for Human and Animal Health, 1015 Arlington Street, Winnipeg, Manitoba R3E 3R2, Canada4
Received 16 July 2008/ Accepted 25 February 2009
We compared dideoxy sequencing of cloned chaperonin-60 universal target (cpn60 UT) amplicons to pyrosequencing of amplicons derived from vaginal microbial communities. In samples pooled from a number of individuals, the pyrosequencing method produced a data set that included virtually all of the sequences that were found within the clone library and revealed an additional level of taxonomic richness. However, the relative abundances of the sequences were different in the two datasets. These observations were expanded and confirmed by the analysis of paired clone library and pyrosequencing datasets from vaginal swabs taken from four individuals. Both for individuals with a normal vaginal microbiota and for those with bacterial vaginosis, the pyrosequencing method revealed a large number of low-abundance taxa that were missed by the clone library approach. In addition, we showed that the pyrosequencing method generates a reproducible profile of microbial community structure in replicate amplifications from the same community. We also compared the taxonomic composition of a vaginal microbial community determined by pyrosequencing of 16S rRNA amplicons to that obtained using cpn60 universal primers. We found that the profiles generated by the two molecular targets were highly similar, with slight differences in the proportional representation of the taxa detected. However, the number of operational taxonomic units was significantly higher in the cpn60 data set, suggesting that the protein-encoding gene provides improved species resolution over the 16S rRNA target. These observations demonstrate that pyrosequencing of cpn60 UT amplicons provides a robust, reliable method for deep sequencing of microbial communities.
* Corresponding author. Mailing address: Agriculture and Agri-Food Canada Saskatoon Research Centre, 107 Science Place, Saskatoon, SK S7N 0X2, Canada. Phone: (306) 956-7653. Fax: (306) 956-7247. E-mail: dumonceauxt{at}agr.gc.ca
Published ahead of print on 6 March 2009.
Supplemental material for this article may be found at http://aem.asm.org/.
Applied and Environmental Microbiology, May 2009, p. 2889-2898, Vol. 75, No. 9
0099-2240/09/$08.00+0 doi:10.1128/AEM.01640-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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