A novel method for determining microflora composition using dynamic phylogenetic analysis of 16S ribosomal RNA deep sequencing data

Abstract

Deep sequencing of the 16S rRNA gene provides a comprehensive view of bacterial communities in a particular environment and has expanded our ability to study the impact of the microflora on human health and disease. Current analysis methods rely on comparisons of the sequences generated with an expanding but limited set of annotated 16S rRNA sequences or phylogenic clustering of sequences based on arbitrary similarity cutoffs. We describe a novel approach to characterize bacterial composition using deep sequencing of 16S rRNA gene. Our method defines operational taxonomic units based on phylogenetic tree reconstruction and dynamic clustering of sequences using solely sequencing data. These OTUs can be used to identify differences in bacteria abundance between environments. This approach can perform better than previous phylogenetic methods and will significantly improve our understanding of the microfloral role on human diseases by providing a comprehensive analysis of the microbial composition from various bacterial communities.

SIGIRR, a Negative Regulator of TLR/IL-1R Signalling Promotes Microbiota Dependent Resistance to Colonization by Enteric Bacterial Pathogens

“…Previous sequencing studies found no overt differences between the microbiota of Sigirr −/− mice and WT mice [32], but considering the susceptibility of Sigirr −/− mice to enteric pathogens, we speculated they might instead carry either fewer total commensal microbes, or harbour a different microbial composition than WT mice. We enumerated the microbiota in the feces of these mice by fluorescence microscopy under baseline conditions, but noted no significant differences in commensal numbers between WT mice and Sigirr −/− mice (Figure 9A).…”

Factoring the intestinal microbiome into the pathogenesis of autoimmune hepatitis

“…Traditional stool culture techniques are limited in assessing the intestinal microbiome mainly because anaerobic organisms are difficult to culture[235] and some microbial species may elude detection by conventional protocols[235,236]. A common method for studying the diversity of the intestinal microflora has been to sequence the 16S ribosomal ribonucleic acid (rRNA) gene[235,237].…”

“…A common method for studying the diversity of the intestinal microflora has been to sequence the 16S ribosomal ribonucleic acid (rRNA) gene[235,237]. The 16S rRNA gene is present in all prokaryotic cells; it has highly variable regions interspersed with highly conserved regions; and its sequences are unique to the major groups of prokaryotic organisms[237].…”

“…The variable regions are amplified by polymerase chain reaction (PCR), and the PCR products are purified for sequencing[81]. Sequencing results are compared to established annotated datasets[235]. The sequencing protocol misidentifies or omits a critical residue in only 1% of procedures[237].…”

“…The sequencing protocol misidentifies or omits a critical residue in only 1% of procedures[237]. Ambiguities resulting from the sequencing determinations are the most common errors, and they may reflect inadequacies in the available datasets[235,237]. Only known bacterial sequences or sequences closely homologous to known bacterial sequences can be analyzed[235].…”