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

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  • James McDonald
  • Niels Larsen
    Danish Genome Institute, Aarhus
  • Andrea Pennington
    University of Liverpool
  • John Connolly
    University of Liverpool
  • Corrin Wallis
    WALTHAM Centre for Pet Nutrition
  • David J. Rooks
    University of Liverpool
  • Neil Hall
    University of Liverpool
  • Alan J. McCarthy
    University of Liverpool
  • Heather E. Allison
    University of Liverpool
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 reversetranscribed
SSU rRNA (RT-SSU rRNA) molecules from the canine oral microbiome and
compared 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 fragmented
RT-SSU rRNA reads produced. Direct RT-SSU rRNA sequencing revealed that
16S 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 molecular
microbial ecology.
Original languageEnglish
Article numbere0157046
JournalPLoS ONE
Issue number6
Publication statusPublished - 8 Jun 2016

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