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. 2012;13 Suppl 8(Suppl 8):S17.
doi: 10.1186/1471-2164-13-S8-S17. Epub 2012 Dec 17.

Species-level classification of the vaginal microbiome

Jennifer M Fettweis  1 , Myrna G Serrano, Nihar U Sheth, Carly M Mayer, Abigail L Glascock, J Paul Brooks, Kimberly K Jefferson; Vaginal Microbiome Consortium (additional members); Gregory A Buck
Affiliations

Species-level classification of the vaginal microbiome

Jennifer M Fettweis et al. BMC Genomics. 2012.

Abstract

Background: The application of next-generation sequencing to the study of the vaginal microbiome is revealing the spectrum of microbial communities that inhabit the human vagina. High-resolution identification of bacterial taxa, minimally to the species level, is necessary to fully understand the association of the vaginal microbiome with bacterial vaginosis, sexually transmitted infections, pregnancy complications, menopause, and other physiological and infectious conditions. However, most current taxonomic assignment strategies based on metagenomic 16S rDNA sequence analysis provide at best a genus-level resolution. While surveys of 16S rRNA gene sequences are common in microbiome studies, few well-curated, body-site-specific reference databases of 16S rRNA gene sequences are available, and no such resource is available for vaginal microbiome studies.

Results: We constructed the Vaginal 16S rDNA Reference Database, a comprehensive and non-redundant database of 16S rDNA reference sequences for bacterial taxa likely to be associated with vaginal health, and we developed STIRRUPS, a new method that employs the USEARCH algorithm with a curated reference database for rapid species-level classification of 16S rDNA partial sequences. The method was applied to two datasets of V1-V3 16S rDNA reads: one generated from a mock community containing DNA from six bacterial strains associated with vaginal health, and a second generated from over 1,000 mid-vaginal samples collected as part of the Vaginal Human Microbiome Project at Virginia Commonwealth University. In both datasets, STIRRUPS, used in conjunction with the Vaginal 16S rDNA Reference Database, classified more than 95% of processed reads to a species-level taxon using a 97% global identity threshold for assignment.

Conclusions: This database and method provide accurate species-level classifications of metagenomic 16S rDNA sequence reads that will be useful for analysis and comparison of microbiome profiles from vaginal samples. STIRRUPS can be used to classify 16S rDNA sequence reads from other ecological niches if an appropriate reference database of 16S rDNA sequences is available.

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Figures

Figure 1
Figure 1
Sequence variation plot of V1-V3 region of the 16S rRNA gene of Lactobacillus, Prevotella, and Staphylococcus reference sequences. Reference sequences from 144 species of Lactobacillus, 43 species of Prevotella, and 37 species of Staphylococcus were included in the analysis (Additional files 1, 2, 3). New reference OTU sequences were excluded from analysis. Smoothed Shannon index values are represented relative to E. coli 16S rRNA gene numbering. Approximate locations of the V1-, V2-, and V3-hypervariable regions are indicated.
Figure 2
Figure 2
Proportion of mid-vaginal reads from Vaginal Human Microbiome Project classified by RDP and STIRRUPS. Blue bars represent the percentage of reads classified by RDP at each taxonomic level. Orange bars represent the additional percentage of reads classified at each level using STIRRUPS in conjunction with the Vaginal 16S rDNA Reference Database. "Species" indicates species-level as described in the Methods.
See this image and copyright information in PMC

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