on is lower than the expected 1% random error rate of pyrosequencing , and very close to the expected 0.25% random error rate of pyrosequencing reads after stringent quality filtering . As a result of this, such low levels of variation are beyond the capacity of commonly available computational methods to separate these variants confidently, resulting in strains that are similar at the 16S rRNA gene level to receive the same taxonomical assignment, or to be collected in one OTU group. Similarly, variation across the sequences we analyzed in this study ranged from 0.2% to 1.66% over the 481 nucleotide-long pyrosequencing reads obtained from the V4–V6 region of the 16S rRNA gene. Therefore, due to the very high similarity among sequences, all would have been considered G. vaginalis, or clustered in one 3% OTU group. In spite of this, we observed a remarkable amount of G. vaginalis diversity, and were able to detect a high degree of correlation between oligotype profiles of many sexual partnerships (see Figure 3).
This relatively large scale study of variation in G. vaginalis 16S rRNA gene sequences supports previous cultivation-based studies that suggest G. vaginalis is sexually transmissible and that male and female partners share similar G. vaginalis strains , . Moreover, results of this study show that the usual approaches used to analyze 454 pyrosequencing data derived from human genitourinary tract samples miss important diversity that may be ecologically, clinically and/or epidemiologically significant.
The UniFrac analysis results appear to suggest that there may be a unique, closely related group of G. vaginalis oligotypes found among GS-defined normal and some GS-defined intermediate women. However, the relatively limited number of GS-defined normal and GS-defined intermediate women included in this study, require these results to be corroborated by additional studies. Nonetheless, results presented here suggest that the oligotyping approach could be used to identify and separate very similar strains at 16S rRNA gene level from high-throughput sequencing data, and explore whether there are specialized types for different ecological niches. Preliminary analysis of Megasphaera spp. has also revealed numerous oligotype distribution profiles among women with GS BV (results not shown), suggesting that applying the method described here to other species that are commonly found in the genitourinary microbiota could yield important new insights. Additionally, consideration should be given to oligotype analyses of other phylogenetically informative genes, such as recA , , in order to explore to which extent the oligotypes at the 16S rRNA gene level are able to predict genomic variation.
In summary, our study describes a novel method for revealing concealed diversity at a very low level of taxonomy by utilizing Shannon entropy to amplify weak signals of subtle but reproducible nucleotide variation within high-throughput sequencing reads. This oligotyping approach can be applied to existing sequence libraries to explore diversity at an ecologically meaningful level and investigate potential ecotypes and their diversity hidden within conventionally defined species.
Phylogenetic distribution of 46 oligotypes. Bars and numbers next to oligotypes indicate how many samples they were present at least once in all samples.
Hierarchical clustering results of samples from male patients. Penile skin (clustering significance: p = 0.011, UniFrac significance: p = 0.001) and urethra (clustering significance: p<0.001, UniFrac significance: p = 0.077) samples were clustered based on the UniFrac distance metric. Red, orange and green colors indicate samples that are sexual partners of GS-defined BV, GS-defined intermediate and GS-defined normal female patients, respectively.
Stacked bar representation of oligotype profiles among couples. While VS labeled bars represent female patients oligotype profile, for every couple MP (penile skin sample) or MU (urethra sample) bars represent male sexual partners oligotype profile. For the sake of compa