Characterising the Canine Oral Microbiome by Direct Sequencing of Reverse-Transcribed rRNA Molecules

PCR amplification and sequencing of phylogenetic markers, primarily Small Sub-Unit ribosomalRNA (SSU rRNA) genes, has been the paradigm for defining the taxonomic composition of microbiomes. However, ‘universal’ SSU rRNA gene PCR primer sets are likely tomiss much of the diversity therein. We sequenced a library comprising purified and reversetranscribedSSU rRNA (RT-SSU rRNA) molecules from the canine oral microbiome andcompared it to a general bacterial 16S rRNA gene PCR amplicon library generated from thesame biological sample. In addition, we have developed BIONmeta, a novel, open-source,computer package for the processing and taxonomic classification of the randomly fragmentedRT-SSU rRNA reads produced. Direct RT-SSU rRNA sequencing revealed that16S rRNA molecules belonging to the bacterial phyla Actinobacteria,Bacteroidetes, Firmicutes,Proteobacteria and Spirochaetes, were most abundant in the canine oral microbiome(92.5% of total bacterial SSU rRNA). The direct rRNA sequencing approach detectedgreater taxonomic diversity (1 additional phylum, 2 classes, 1 order, 10 families and 61 genera)when compared with general bacterial 16S rRNA amplicons from the same sample,simultaneously provided SSU rRNA gene inventories of Bacteria, Archaea and Eukarya,and detected significant numbers of sequences not recognised by ‘universal’ primer sets.Proteobacteria and Spirochaetes were found to be under-represented by PCR-based analysisof the microbiome, and this was due to primer mismatches and taxon-specific variationsin amplification efficiency, validated by qPCR analysis of 16S rRNA amplicons from a mockcommunity. This demonstrated the veracity of direct RT-SSU rRNA sequencing for molecularmicrobial ecology.