Pyrosequencing of the Chaperonin-60 Universal Target as a Tool for Determining Microbial Community Composition

Abstract

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.

A ‘universal’ type II chaperonin PCR detection system for the investigation of Archaea in complex microbial communities

“…The strength of the thermosome gene as a target is that it combines universality (like the 16S rRNA gene) with the greater sequence diversity associated with a protein-encoding gene (like the mcrA gene). The benefits of targeting a protein-encoding gene have been seen in bacteria, where the cpn60 (type I chaperonin) UT has advantages over 16S rRNA gene in terms of discrimination of closely related taxa and bioinformatics (Schellenberg et al, 2009).…”

“…A combination of BLAST and Smith-Waterman alignments (watered-BLAST; Schellenberg et al, 2009) was used to identify the best match (nearest neighbour) for each individual sequence read from an appropriate reference sequence collection (Supplementary Table 1). Results were filtered to remove matches shorter than 100 bp to ensure reliable identifications.…”

“…In Archaea, organisms possess one to three parologous thermosome genes, giving rise to a, b and g subunits. The thermosome gene is an appealing target as its bacterial homologue, cpn60, has been demonstrated to be an excellent target for species detection, identification and quantification of individual bacterial species and strains as well as the metagenomic characterization of complex microbial communities (Hill et al, 2005;Dumonceaux et al, 2006b;Schellenberg et al, 2009). In addition, the infrastructure needed to translate the thermosome sequence into a useful identification tool is already in place since thermosome sequences have been included in the cpnDB database since its creation (Hill et al, 2004).…”

“…While the slow rate of evolution allows for broad taxonomic comparison, this same characteristic results in nearly identical sequences for closely related organisms, reducing the resolution of phylogenetic trees and making differentiation of these taxa difficult (Wang et al, 2007;Schellenberg et al, 2009;Weng et al, 2009). As well, the regular occurrence of insertions and deletions (indels) in rRNA genes has resulted in challenges for multiple sequence alignments, the first step in phylogenetic analysis (DeSantis et al, 2006).…”

“…Of these, the type I chaperonin gene, cpn60 (also known as hsp60 or groEL) is the most developed alternative. It is the only target other than 16S rRNA gene that can be accessed with 'universal' PCR primers and a curated sequence database, cpnDB, is available (http://www.cpndb.ca; Hill et al, 2004Hill et al, , 2006bSchellenberg et al, 2009). The cpn60 gene provides greater discriminating power than 16S rRNA gene for closely related taxa and the uniform size and sequence heterogeneity of the cpn60 'universal target' (UT) simplify sequence comparisons and other bioinformatics tasks.…”