High-resolution Amplicon Sequencing Detects a Personalized, Stable Oral Microbiome

Objectives: 16S rRNA gene sequencing has elucidated species-level microbial community structures. However, many important questions require understanding of community and population structures at the level of strains. Metagenomic approaches are expensive, and it is difficult to resolve strains from complex communities containing multiple representatives of closely related microbes. We developed an efficient sub-species-level, high-resolution approach by coupling targeted sequencing of the ribosomal intergenic spacer region (ISR) with high-resolution bioinformatic processing. We then used the approach to track oral community composition over time.

Methods: Subgingival plaque samples were collected at 6 time points over 1 year from 5 healthy subjects. Amplicon libraries targeting the 16S gene and the adjacent ISR were sequenced using Illumina MiSeq. 16S reads were processed both by mapping against the CORE database and by de novo binning using the high-resolution DADA2 pipeline. ISR reads were processed using DADA2 and assigned to species using an ISR-database constructed by extracting relevant sequences from publicly available genomes.

Results: Bray-Curtis dissimilarity analysis showed that each subject exhibited a distinct subspecies community profile. Drift over time in the microbial profiles of every subject was observed, yet intra-subject distances were consistently smaller, even over 1 year, than inter-subject distances. Most common oral species could be resolved into a number of ISR-variants or ISR-type strains. Both shared variants and variants unique to individual subjects were observed. For example, in the Streptococcus mitis group, Haemophilus parainfluenza and Granulicatella adiacens, 26 out of 51, 25 out of 41 and 15 out of 38 iStrains were observed in more than one subject, respectively.

Conclusions: Our high-resolution ISR amplicon sequencing and processing approach provides an effective high-throughput tool for studying microbial community composition at a resolution beyond species level. Oral ISR-type strain profiles for each subject were distinct, even samples taken one year apart could be distinguished from all other subjects. This efficient approach will make large-scale tracking of bacterial strains in comprehensive studies possible.