Using high throughput sequencing to explore the biodiversity in oral bacterial communities

Area of Science:

• Microbiology
• Genomics
• Bioinformatics

Background:

• High throughput sequencing of 16S ribosomal RNA (rRNA) gene amplicons is a cost-effective method for characterizing oral bacterial communities.
• Understanding technique-associated limitations and intrinsic variability is crucial before large-scale oral microbiome studies.

Purpose of the Study:

• Evaluate bias in species representation using an in vitro mock community.
• Characterize oral bacterial communities in saliva and buccal mucosa of healthy subjects.
• Investigate the power of high throughput sequencing for revealing oral diversity and biogeography patterns.

Main Methods:

• Assembled an in vitro mock community of oral bacteria for bias evaluation.
• Sequenced 16S rRNA gene amplicons from saliva and buccal mucosa samples of five healthy individuals.
• Analyzed operational taxonomic units (OTUs) and community structure.

Main Results:

• Mock community analysis revealed primer and DNA isolation biases, leading to overestimation of diversity, which decreased after removing singleton OTUs.
• Sequencing identified 455 OTUs in salivary and mucosal communities, with only 78 shared across all subjects.
• Inter-subject variability in community structure was lower than site-specific differences (saliva vs. mucosa), largely due to Streptococcus mitis and Gemella haemolysans abundance on mucosa.

Conclusions:

• High throughput sequencing, despite technical challenges, is powerful for evaluating oral diversity and biogeography.
• Incomplete richness coverage necessitates comparing community structure over membership for robust analysis.
• An experimental and data analysis framework is provided to aid pyrosequencing-based oral microbiome studies.