An accurate and efficient experimental approach for characterization of the complex oral microbiota

Abstract

BackgroundCurrently, taxonomic interrogation of microbiota is based on amplification of 16S rRNA gene sequences in clinical and scientific settings. Accurate evaluation of the microbiota depends heavily on the primers used, and genus/species resolution bias can arise with amplification of non-representative genomic regions. The latest Illumina MiSeq sequencing chemistry has extended the read length to 300 bp, enabling deep profiling of large number of samples in a single paired-end reaction at a fraction of the cost. An increasingly large number of researchers have adopted this technology for various microbiome studies targeting the 16S rRNA V3–V4 hypervariable region.ResultsTo expand the applicability of this powerful platform for further descriptive and functional microbiome studies, we standardized and tested an efficient, reliable, and straightforward workflow for the amplification, library construction, and sequencing of the 16S V1–V3 hypervariable region using the new 2 × 300 MiSeq platform. Our analysis involved 11 subgingival plaque samples from diabetic and non-diabetic human subjects suffering from periodontitis. The efficiency and reliability of our experimental protocol was compared to 16S V3–V4 sequencing data from the same samples. Comparisons were based on measures of observed taxonomic richness and species evenness, along with Procrustes analyses using beta(β)-diversity distance metrics. As an experimental control, we also analyzed a total of eight technical replicates for the V1–V3 and V3–V4 regions from a synthetic community with known bacterial species operon counts. We show that our experimental protocol accurately measures true bacterial community composition. Procrustes analyses based on unweighted UniFrac β-diversity metrics depicted significant correlation between oral bacterial composition for the V1–V3 and V3–V4 regions. However, measures of phylotype richness were higher for the V1–V3 region, suggesting that V1–V3 offers a deeper assessment of population diversity and community ecology for the complex oral microbiota.ConclusionThis study provides researchers with valuable experimental evidence for the selection of appropriate 16S amplicons for future human oral microbiome studies. We expect that the tested 16S V1–V3 framework will be widely applicable to other types of microbiota, allowing robust, time-efficient, and inexpensive examination of thousands of samples for population, phylogenetic, and functional crossectional and longitutidal studies.

The Denture-Associated Oral Microbiome in Health and Stomatitis

“…Furthermore, unlike our results detailed above, a recently published sequencing study (12) described a significant difference between the bacterial community compositions found on denture surfaces and those on remaining teeth. The apparent discrepancies between our findings and previous 16S rRNA gene-based sequencing studies can be due to many factors, ranging from geographical differences between patient populations to sample collection, sequencing parameters (choice of 16S rRNA target region, the sequencing platform used, available read length, and sequencing depth, among others), or DNA extraction and PCR protocols (37). …”

Effects of Specimen Collection Methodologies and Storage Conditions on the Short-Term Stability of Oral Microbiome Taxonomy

“…Although the number of adult participants was small, the number of samples collected per individual was sufficient to determine that these factors have minimal impact. This is consistent with other similar studies conducted using different microbiomes (35)(36)(37).…”

Microbiota profile is different for early and invasive colorectal cancer and is consistent throughout the colon

“…In this research we have utilised the eHOMD database for OTU picking instead of most commonly used SILVA and Greengenes databases. As eHOMD is a more recent database and focused on where originally Fn was found (oral microbiota), we understand that this would provide more accurate results for our main outcome.…”