Abstract
BackgroundThe 16S rRNA gene is the gold standard in molecular surveys of bacterial and archaeal diversity, but it has the disadvantages that it is often multiple-copy, has little resolution below the species level and cannot be readily interpreted in an evolutionary framework. We compared the 16S rRNA marker with the single-copy, protein-coding rpoB marker by amplifying and sequencing both from a single soil sample. Because the higher genetic resolution of the rpoB gene prohibits its use as a universal marker, we employed consensus-degenerate primers targeting the Proteobacteria.Methodology/Principal FindingsPyrosequencing can be problematic because of the poor resolution of homopolymer runs. As these erroneous runs disrupt the reading frame of protein-coding sequences, removal of sequences containing nonsense mutations was found to be a valuable filter in addition to flowgram-based denoising. Although both markers gave similar estimates of total diversity, the rpoB marker revealed more species, requiring an order of magnitude fewer reads to obtain 90% of the true diversity. The application of population genetic methods was demonstrated on a particularly abundant sequence cluster.Conclusions/SignificanceThe rpoB marker can be a complement to the 16S rRNA marker for high throughput microbial diversity studies focusing on specific taxonomic groups. Additional error filtering is possible and tests for recombination or selection can be employed.
Highlights
The small subunit ribosomal RNA gene is the recognized gold standard for estimating the phylogenetic diversity in microbial communities (e.g. [1,2,3])
This is further complicated by the fact that sequence variation between the different 16S rRNA gene copies present exists in some genomes [11,12]). rpoB typically occurs in a single copy [9]
We examined the performance of both markers with respect to quantifying diversity of the proteobacterial fraction of the community
Summary
The small subunit ribosomal RNA gene is the recognized gold standard for estimating the phylogenetic diversity in microbial communities (e.g. [1,2,3]). Since most bacterial genomes contain multiple copies of the 16S rRNA gene, and copy number varies per species, extrapolation of relative abundances from gene recovery frequencies is seriously impaired. This is further complicated by the fact that sequence variation between the different 16S rRNA gene copies present exists in some genomes [11,12]). The 16S rRNA gene is the gold standard in molecular surveys of bacterial and archaeal diversity, but it has the disadvantages that it is often multiple-copy, has little resolution below the species level and cannot be readily interpreted in an evolutionary framework. Because the higher genetic resolution of the rpoB gene prohibits its use as a universal marker, we employed consensus-degenerate primers targeting the Proteobacteria
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