An optimized 16S rRNA sequencing protocol for vaginal microbiome to avoid biased abundance estimation

We applied three 16S rRNA sequencing protocols on vaginal microbiome samples, to evaluate whether they produce unbiased estimation of vaginal microbiome composition. We modified the 27F primer (hereafter denoted as 27F). Using vaginal samples from 28 healthy women and 10 women with bacterial vaginosis, we sequenced three 16S rRNA sequencing protocols, i.e., 27F-338R, 27F-338R and 341F-806R protocols, naming after their PCR primer sets, to test whether the sequencing results are consistent with the clinical diagnostics, morphology and qPCR results. First, the 27F primer would not align with Gardnerlla vaginalis very well, leading to poor amplification of such species. By modifying the primer sequences, the modified 27F primer (27F) was able to amplify Gardnerlla vaginalis very well. Second, the DNA sequence of characteristic species Lactobacillus crispatus is identical with Lactobacillus garrinarum, leading to biased estimation of abundance of Lactobacillus crispatus when using V3-V4 as PCR target region; in contrast, such bias did not occur when using V1-V2 as a target region. Third, optimized 27F-338R avoided above-mentioned biases and restored the well-established community state types (CSTs) clustering. ImportanceVaginal microbiome has profound effects on the health of women and their newborns. Our study found that two well-established 16S rDNA sequencing protocols led to systementical biased estimation of characteristic species of vaginal microbiome. Subsequent analysis proved that the PCR primer fetching efficacy and target region identity were major contributor for such bias. With carefully selected target region and optimized PCR primer set, we were able to eliminate such biases and provide accurate estimation of vaginal microbiome, which showed high consistency with clinical diagnostics. We modified the 27F primer (27F). Using the optimized PCR primer set of 27F and 338R to target the V1-V2 hyper-variable region, our 16S rRNA sequencing correctly evaluate the composition of vaginal microbiome.