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

Abstract

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.

The Gut Microbiota: Ecology and Function

“…Phylogenetic representation in the distal SI also increases and includes those genera indicated previously, plus members of genera within the Clostridiales and Bacteriodetes families (Hill et al, 2004;Richards et al, 2005). Complexity of the microbial community is greatest in the large intestine, where the density increases to 10 10 to 10 12 CFU per gram and the community is dominated by gram-positive genera, including Clostridium, Bacillus, Ruminococcus, and Fusobacterium (Hill et al, 2002;Leser et al, 2002). Variation within bacterial species among locations in the GI tract 12 also substantially contributes to overall gut microbial diversity and function (Dixit et al, 2004).…”

Culture clash: challenging the dogma of microbial diversity

“…Others have reported that early and key steps in the ribosomal approach such as those pertaining to PCR conditions and template to product ratios affect the composition of the resultant clone libraries (Mathieu-Daudé et al, 1996;Suzuki and Giovannoni, 1996;Polz and Cavanaugh, 1998;Bonnet et al, 2002;Hongoh et al, 2003). In addition, high DNA sequencing costs until recently meant analysis of small sequences and small, unrepresentative libraries was tacitly accepted (Hill et al, 2002). It remains to be seen if high-throughput cultivation and sequencing techniques will enable 'molecular methods' to fulfill the promise of detecting all microbes in a community, or if we really never will know the true extent of microbial diversity (Curtis et al, 2002).…”

Apparent absorption of methionine and 2-hydroxy-4-methylthiobutanoic acid from gastrointestinal tract of conventional and gnotobiotic pigs

“…Briefly, after overnight culture from frozen stocks in TS broth, bacterial cells were harvested by centrifugation and genomic DNA was extracted with phenol-chloroform-isoamyl alcohol according to previously described methods (Dumonceaux et al, 2006). The cpn60 UT was amplified by PCR using 1 ml of extracted DNA, 0.5 U of Taq polymerase, 50 mM MgCl 2 , 10 mM of dNTPs (deoxynucleotide triphosphates) and 0.375 mM for each of the degenerate primers H729 and H730 (Hill et al, 2002). Reactions were subject to 958C for 3 min, followed by 40 cycles of 1 min at 958C, 1 min at 468C, 1 min at 728C and a terminal 5 min extension at 728C.…”