Complementing 16S rRNA gene amplicon sequencing with estimates of total bacterial load to infer absolute species concentrations in the vaginal microbiome

Abstract

Whereas 16S rRNA gene amplicon sequencing quantifies relative abundances of bacterial taxa, variation in total bacterial load between samples restricts its ability to reflect absolute concentration of individual species. Quantitative PCR (qPCR) can quantify individual species, but it is not practical to develop a suite of qPCR assays for every bacterium present in a diverse sample. We analyzed 1320 samples from 20 women with a history of frequent bacterial vaginosis, who self-collected vaginal swabs daily over 60 days. We inferred bacterial concentrations by taking the product of species relative abundance (assessed by 16S rRNA gene amplicon sequencing) and total bacterial load (measured by broad-range 16S rRNA gene qPCR). Log10-converted inferred concentrations correlated with targeted qPCR (r = 0. 935, p0.5 log10 occurred when relative abundance was 10%, inferred concentrations are reliable proxies for targeted qPCR. However, targeted qPCR is required to capture bacteria at low relative abundance, particularly with BV-associated bacteria during the early onset of bacterial vaginosis.