Selection of validated hypervariable regions is crucial in 16S-based microbiota studies of the female genital tract

NGS-based partial 16S rRNA gene amplicon sequencing methods are extensively applied in studies of the human microbiota, but carry drawbacks that are critical to consider when interpreting results, including differences in outcome based on the hypervariable region(s) considered. Here, we show that primers spanning the V3/V4 region identify a greater number of taxa in the vaginal microbiota than those spanning the V1/V2 region. In particular, taxa such as Gardnerella vaginalis, Bifidobacterium bifidum and Chlamydia trachomatis, all species influencing vaginal health and disease, are not represented in V1/V2-based community profiles. Accordingly, missing or underestimating the frequency of these species overestimates the abundance of other taxa and fails to correctly assess the bacterial diversity in the urogenital tract. We elaborate that covering these taxa using the V3/V4 region leads to profound changes in the assignment of community state types. Altogether, we show that the choice of primers used for studying the vaginal microbiota has deep implications on the biological evaluation of the results.