Analysis of microbial diversity by pyrosequencing the small-subunit ribosomal RNA without PCR amplification.

To avoid the biases associated with PCR amplification in analysis of microbial communities, a new method has been tested for direct sequencing of the cDNA of full-length Small-subunit Ribosomal RNA withOut specific PCR amplification (SROP). In silico analysis of the SROP method demonstrated that more than 99 % of the SROP sequences could be correctly annotated. Two environmental samples (activated sludge and anaerobic sludge) with complex microbial communities were used for comparison in this study. The SROP results demonstrated that the families Rhodocyclaceae and Nitrosomonadaceae in activated sludge and the phyla Synergistetes and Spirochaetes in anaerobic sludge were underestimated by PCR-based detection. One third of the 16S ribosomal RNA (rRNA) sequences obtained by the SROP method covered the V3 amplicon region, and they are suitable for phylogenetic and diversity index analyses. The microbial diversity index calculated from the rRNA sequences by the SROP was much higher than that calculated by conventional PCR, particularly for the anaerobic sludge. The metatranscriptome-based SROP method will contribute to our better understanding of the diversity of complex microbial communities.