 representing each oligotype was tallied for each sample to generate a 46-dimensional feature vector where each component of the vector reflected the percent abundance of the corresponding oligotype within the given sample. Pearson correlation analysis results are listed in Table 2.
Phylogenetic analysis of oligotypes and UniFrac clustering
Phylogenetic relationships among the oligotypes were assessed with Bayesian inference using MrBayes (version 3.1.2, http://mrbayes.sourceforge.net/) , . Analysis was initiated with random starting trees with representative sequences for each oligotype, and posterior probabilities were determined from two independent runs of one million generations of Markov chain Monte Carlo simulations, from which tree topologies were sampled every 100 generations. After discarding the first 25% of resulting trees, a consensus phylogenetic tree of oligotypes was estimated from remaining generations (Figure S1). The resulting tree was used as a common phylogeny to perform UniFrac analysis . Hierarchical clustering of oligotypes in vaginal (Figure 2), and penile skin and urethra samples (Figure S2) was performed based on distance matrices generated by the unweighted UniFrac analysis. Tree topology of the phylogenetic analysis and clustering results were visualized using the Interactive Tree of Life .
Samples were clustered (clustering significance: p<0.001, UniFrac significance: p = 0.016) based on the UniFrac distance metric. Red, orange and green colors indicate samples from BV, intermediate and normal Nugent categories, respectively.
The generation of oligotype profiles by merging nucleotides from variable locations of G. vaginalis tag sequences that are revealed by Shannon entropy analysis (Figure 1) made it possible to compare samples to each other based on their G. vaginalis oligotype compositions. This analysis showed extensive diversity within G. vaginalis sequences from different samples, as well as significant correlations between the oligotype profiles of many couples. The composition of G. vaginalis oligotypes in vaginal samples of 24 of 44 women, whose partners also harbored at least one G. vaginalis sequence, were significantly correlated (r≥0.9, p<0.001) with either the penile skin, or urethral, or both samples from their sexual partners (Table 2). Significant correlation in G. vaginalis oligotypes was observed between vaginal and penile skin samples in 19 couples, while for vaginal and urethral samples of only 12 couples had correlation values above 0.9. In 8 couples, there was reduced, but nonetheless high degree of correlation (r≥0.5, p<0.001) between the vaginal and either the penile skin or the urethral samples. In 12 couple no correlation was found between partners (r<0.5). Figure 3 illustrates seven couples whose G. vaginalis compositions are highly correlated (see Figure S3 for stacked bar chart comparison of all samples). Correlation levels between partners did not appear to vary significantly by GS classification, although, the total number of couples in the intermediate and normal categories is small and the total number of G. vaginalis sequences in normal couples is low (Table 1).
Different colors in the pie charts correspond to different oligotypes. In every set, the pie chart on the left represents the sample collected from the female patient.
The oligotype TCCCTCGA was the most abundant overall and it was observed in most of the samples (Table S2). It was the dominant oligotype of 24 of 48 vaginal samples. The TCCCACAG oligotype was the dominant oligotype in 10 vaginal samples. While the TTTTACGA, TTCTACGA and TTCCTCGA were dominant in 3 vaginal samples each, oligotypes TTTTATGA, TTCTTCGA and TCTCACGA were dominant in one vaginal sample each. A complete list of oligotype distribution across genders, anatomical sampling sites, and GS-defined BV and GS-defined normal microbiota is given in Table S2.
UniFrac ,  is a computational method used to compare microbial samples to each other based on their composition with respect to a common phylogeny. After computing a phylogenetic