Assessment of in Vitro and in Silico Protocols for Sequence-Based Characterization of the Human Vaginal Microbiome

The vaginal microbiome has been connected to a wide range of health outcomes. This has led to a thriving research environment, but also to the use of conflicting methodologies to study its microbial composition. Here we systematically assess best practices for the sequencing-based characterization of the human vaginal microbiome. As far as 16S rRNA gene sequencing is concerned, the V1-V3 region has the best theoretical properties, but limitations of current sequencing technologies mean that the V3-V4 region performs equally well. Both of these approaches present very good agreement with qPCR quantification of key taxa, provided an appropriate bioinformatic pipeline is used. Shotgun metagenomic sequencing presents an interesting alternative to 16S amplification and sequencing, but it is not without its challenges. We have assessed different tools for the removal of host reads and the taxonomic annotation of metagenomic reads, including a new, easy-to-build and – use, reference database of vaginal taxa. This strategy performed as well as the best performing previously published strategies. Despite the many advantages of shotgun sequencing none of the shotgun approaches assessed here had as good agreement with the qPCR data as 16S rRNA gene sequencing. Importance The vaginal microbiome has been connected to a wide range of health outcomes, from susceptibility to sexually transmitted infections to gynecological cancers and pregnancy outcomes. This has led to a thriving research environment, but also to conflicting available methodologies, including many studies that do not report their molecular biological and bioinformatic methods in sufficient detail for them to be considered reproducible. This can lead to conflicting messages and delay progress from descriptive to intervention studies. By systematically assessing best practices for the characterization of the human vaginal microbiome, this study will enable past studies to be assessed more critically and assist future studies in the selection of appropriate methods for their specific research questions.