Characterising the Canine Oral Microbiome by Direct Sequencing of Reverse-Transcribed rRNA Molecules.
PCR amplification and sequencing of phylogenetic markers, primarily Small Sub-Unit ribosomal RNA (SSU rRNA) genes, has been the paradigm for defining the taxonomic composition of microbiomes. However, 'universal' SSU rRNA gene PCR primer sets are likely to miss much of the diversity therein. We sequenced a library comprising purified and reverse-transcribed 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 the same 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 detected greater 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 analysis of the microbiome, and this was due to primer mismatches and taxon-specific variations in amplification efficiency, validated by qPCR analysis of 16S rRNA amplicons from a mock community. This demonstrated the veracity of direct RT-SSU rRNA sequencing for molecular microbial ecology.
School of Biological Sciences, Bangor University, Deiniol Road, Bangor, Gwynedd, LL57 2UW, United Kingdom.
James E
JE
McDonald
Danish Genome Institute, Skt. Lucas Kirkeplads 8, 8000, Aarhus C, Denmark.
Niels
N
Larsen
Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool, L69 7ZB, United Kingdom.
Andrea
A
Pennington
Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool, L69 7ZB, United Kingdom.
John
J
Connolly
WALTHAM® Centre for Pet Nutrition, Freeby Lane, Waltham-on-the-Wolds, Melton Mowbray, LE14 4RT, United Kingdom.
Corrin
C
Wallis
Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool, L69 7ZB, United Kingdom.
David J
DJ
Rooks
Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool, L69 7ZB, United Kingdom.
Neil
N
Hall
Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool, L69 7ZB, United Kingdom.
Alan J
AJ
McCarthy
Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool, L69 7ZB, United Kingdom.
Heather E
HE
Allison
D000818:Animals; D001419:Bacteria / Q000145:classification / Q000235:genetics; D004285:Dogs; D059014:High-Throughput Nucleotide Sequencing; D064307:Microbiota / Q000235:genetics*; D009055:Mouth / Q000382:microbiology*; D012329:RNA, Bacterial / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*
D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S