Shallow shotgun metagenomic sequencing of vaginal microbiomes with the Oxford Nanopore technology enables the reliable determination of vaginal community state types and broad community structures.

Background: The vaginal microbiome plays an important role in female health; it is associated with reproductive success, susceptibility to sexually transmitted infections, and, importantly, the most prevalent vaginal condition in reproduction-age women, bacterial vaginosis (BV). Traditionally, 16S rRNA gene sequencing-based approaches have been used to characterize the composition of vaginal microbiomes, but shallow shotgun metagenomic sequencing (SMS) approaches, in particular when implemented with the Oxford Nanopore Technologies, have important potential advantages with respect to cost effectiveness, speed of data generation, and the availability of flexible multiplexing schemes.

Results: Based on a study cohort of n = 52 women, of which 23 were diagnosed with BV, we evaluated the applicability of Nanopore-based SMS for the characterization of vaginal microbiomes in direct comparison to Illumina 16S-based sequencing. We observed perfect agreement between the two approaches with respect to detecting the dominance of individual samples by either Lactobacilli, vaginosis-associated, or other taxa; very high concordance (92%) with respect to community state type (CST) classification; and a high degree of concordance with respect to the overall clustering structures of the sequenced microbiomes. Comparing the inferred abundances of individual species in individual samples, we observed significant differences (Wilcoxon signed-rank test p < 0.05) between the two approaches for 12 of the 20 species most abundant in our cohort, indicating differences in the fine-scale characterization of vaginal microbiomes. Higher overall abundance of Gardnerella vaginalis, associated with an increased number of CST IV detections, in the Nanopore shallow SMS data indicated potentially increased sensitivity of this approach to dysbiotic states of the vaginal microbiome. Nanopore shallow SMS also enabled the methylation-based quantification of different human cell types in the characterized samples as well as the detection of non-prokaryotic species, including Lactobacillus phage and Candida albicans in study participants with microscopically detected Candida. One important potential limitation of the evaluated Nanopore-based SMS approach was marked variation in sequencing yields.

Conclusion: Our study demonstrated the successful application and potential advantages of Nanopore-based shallow SMS for the characterization of vaginal microbiomes and paves the way for its application in larger-scale research or diagnostic settings.