Strain-Level Profiling of Oral Microbiota with Targeted Sequencing

Targeted sequencing of one or more regions of the bacterial 16S rRNA gene fragment has emerged as a gold standard for investigating taxonomic diversity in complex microbial communities, such as those found in the oral cavity. While this approach is useful for identifying bacteria up to genus level, its ability to distinguish between many closely related oral species, or explore strain-level variations within each species, is very limited. Here we present an approach based on targeted sequencing the 16S-23S Intergenic Spacer Region (ISR) in the bacterial ribosomal operon for taxonomic characterization of microbial communities at a subspecies or strain level. This approach retains the advantages of 16S-based methods, such as easy library preparation, high throughput, short amplicon sizes, and low cost of sequencing, while providing subspecies-level resolution as a result of naturally higher genetic diversity present in the ISR compared to the 16S hypervariable regions. These advantages make it an excellent tool for high-resolution oral microbiota characterization.