Abstract
BackgroundSequencing of the 16S rRNA gene has been the standard for studying the composition of microbial communities. While it allows identification of bacteria at the level of species, this method does not usually provide sufficient information to resolve communities at the sub-species level. Species-level resolution is not adequate for studies of transmission or stability or for exploring subspecies variation in disease association. Strain level analysis using whole metagenome shotgun sequencing has significant limitations that can make it unsuitable for large-scale studies. Achieving sufficient depth of sequencing can be cost-prohibitive, and even with adequate coverage, deconvoluting complex communities such as the oral microbiota is computationally very challenging. Thus, there is a need for high-resolution, yet cost-effective, high-throughput methods for characterizing microbial communities.ResultsSignificant improvement in resolution for amplicon-based bacterial community analysis was achieved by combining amplicon sequencing of a high-diversity marker gene, the ribosomal 16-23S intergenic spacer region (ISR), with a probabilistic error modeling based denoising algorithm, DADA2. The resolving power of this new approach was compared to that of both standard and high-resolution 16S-based approaches using a set of longitudinal subgingival plaque samples. The ISR strategy resulted in a 5.2-fold increase in community resolution compared to reference-based 16S rRNA gene analysis and showed 100% accuracy in predicting the correct source of a clinical sample. Individuals’ microbial communities were highly personalized, and although they exhibited some drift in membership and levels over time, that difference was always smaller than the differences between any two subjects, even after 1 year. The construction of an ISR database from publicly available genomic sequences allowed us to explore genomic variation within species, resulting in the identification of multiple variants of the ISR for most species.ConclusionsThe ISR approach resulted in significantly improved resolution of communities and revealed a highly personalized human oral microbiota that was stable over 1 year. Multiple ISR types were observed for all species examined, demonstrating a high level of subspecies variation in the oral microbiota. The approach is high-throughput, high-resolution yet cost-effective, allowing subspecies-level community fingerprinting at a cost comparable to that of 16S rRNA gene amplicon sequencing. It will be useful for a range of applications that require high-resolution identification of organisms, including microbial tracking, community fingerprinting, and potentially for identification of virulence-associated strains.
Highlights
Sequencing of the 16S rRNA gene has been the standard for studying the composition of microbial communities
Higher diversity in the intergenic spacer region (ISR) compared to 16S V1-V3 We evaluated the potential of the ribosomal 16-23S ISR for exploring diversity within species by determining how much variation was present among oral bacteria within the ISR compared to the 16S V1-V3 hypervariable region
These alignments indicated, as expected, the ISR could be targeted as a higher resolution marker for amplicon sequencing of the oral microbiota
Summary
Sequencing of the 16S rRNA gene has been the standard for studying the composition of microbial communities. The technique offers several major advantages, such as high throughput, established bioinformatic pipelines and reference databases, and low per sample cost [1]. Use of this approach has revealed the remarkably diverse character of the microbiota [2] and significantly advanced our understanding of its role in human health and disease in multiple body sites [3,4,5,6,7,8,9,10]. More recent evidence from comparative genomics has shown a significant amount of variation within the genomes of different strains of the same species [11]. These genetic variations often lead to significant phenotypic differences among strains [12], which can result in varying degrees of pathogenicity [13]
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