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Applied and Environmental Microbiology, June 2002, p. 3055-3066, Vol. 68, No. 6
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.6.3055-3066.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Extensive Profiling of a Complex Microbial Community by High-Throughput Sequencing

Janet E. Hill,1 Robyn P. Seipp,1 Martin Betts,1 Lindsay Hawkins,1 Andrew G. Van Kessel,2 William L. Crosby,1,3 and Sean M. Hemmingsen1,4*

National Research Council Plant Biotechnology Institute,1 Department of Poultry and Animal Sciences,2 Department of Biochemistry,3 Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Canada4

Received 10 January 2002/ Accepted 12 March 2002

Complex microbial communities remain poorly characterized despite their ubiquity and importance to human and animal health, agriculture, and industry. Attempts to describe microbial communities by either traditional microbiological methods or molecular methods have been limited in both scale and precision. The availability of genomics technologies offers an unprecedented opportunity to conduct more comprehensive characterizations of microbial communities. Here we describe the application of an established molecular diagnostic method based on the chaperonin-60 sequence, in combination with high-throughput sequencing, to the profiling of a microbial community: the pig intestinal microbial community. Four libraries of cloned cpn60 sequences were generated by two genomic DNA extraction procedures in combination with two PCR protocols. A total of 1,125 cloned cpn60 sequences from the four libraries were sequenced. Among the 1,125 cloned cpn60 sequences, we identified 398 different nucleotide sequences encoding 280 unique peptide sequences. Pairwise comparisons of the 398 unique nucleotide sequences revealed a high degree of sequence diversity within the library. Identification of the likely taxonomic origins of cloned sequences ranged from imprecise, with clones assigned to a taxonomic subclass, to precise, for cloned sequences with 100% DNA sequence identity with a species in our reference database. The compositions of the four libraries were compared and differences related to library construction parameters were observed. Our results indicate that this method is an alternative to 16S rRNA sequence-based studies which can be scaled up for the purpose of performing a potentially comprehensive assessment of a given microbial community or for comparative studies.

* Corresponding author. Mailing address: NRC Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, S7N 0W9, Canada. Phone: (306) 975-5242. Fax: (306) 975-4839. E-mail: hemmings{at}cbrpbi.pbi.nrc.ca.

Applied and Environmental Microbiology, June 2002, p. 3055-3066, Vol. 68, No. 6
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.6.3055-3066.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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