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

Abstract

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 assessed 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 performed best in silico, but limitations of current sequencing technologies meant that the V3-V4 region performed equally well. Both approaches presented very good agreement with qPCR quantification of key taxa, provided that an appropriate bioinformatic pipeline was used. Shotgun metagenomic sequencing presents an interesting alternative to 16S rRNA gene amplification and sequencing but requires deeper sequencing and more bioinformatic expertise and infrastructure. We 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 curated database performed as well as the best-performing previously published strategies. Despite the many advantages of shotgun sequencing, none of the shotgun approaches assessed here agreed with the qPCR data as well as the 16S rRNA gene sequencing. IMPORTANCE The vaginal microbiome has been connected to various aspects of host health, including susceptibility to sexually transmitted infections as well as 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 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.

A comparison of approaches to scaffolding multiple regions along the 16S rRNA gene for improved resolution

“…First, although the authors of the SMURF algorithm claim species level resolution, this is obviously limited by database resolution. With specialized, well curated databases like the Optivag database or Human Oral Microbiome Database, species level resolution is achievable and trust-worthy [19,40,41]. However, more general databases like the Silva database may not provide accurate annotation at lower taxonomic levels, especially because Silva does not curate species assignments [23].…”

“…Real Data We used a set of 24 vaginal samples (8 individuals with 3 replicates) which have been previously described [19] (Table S1; Supplemental Methods). The vaginal samples were compared to the Optivag 16S rRNA database (v0.1) [19]. This curated, vagina-specific database provides accurate species level assignments.…”

“…We compared a current approach -ASVs drawn from a single region to samples reconstructed using OTUs, ASVs from both V13 and V34 regions, and Sidle annotated with Optivag database. Optivag is an environment specific, manually curated database, designed to allow accurate species level annotation in vaginal communities [19]. We next looked at the taxonomic composition of the individuals using species-level data.…”

Metagenome-validated Parallel Amplicon Sequencing and Text Mining-based Annotations for Simultaneous Profiling of Bacteria and Fungi: Vaginal Microbiome and Mycobiota in Healthy Women

“…For bacteria, we used the same 16S rRNA gene primers for V3-V4 region as before, speci cally 341F 5′-CCTACGGGNGGCWGCAG-3′ and 785Rev 5′-GACTACHVGGGTATCTAATCC-3′ [19,20]. These primers have been validated to provide high taxon coverage for vaginal bacteria [21]. The reaction comprised of 5 ng/ µl template, 1X Phusion® Master Mix (ThermoFisher, catalog number: F-531L), 0.375 µM V3-V4 locus speci c primers and DMSO.…”

The Healthy Female Microbiome Across Body Sites: Effect of Hormonal Contraceptives and the Menstrual Cycle

“…Similarly, vaginal samples were reannotated with the same tools and the OptiVag database [42]. Due to the uneven amount of annotated bacterial reads generated, each of these samples was filtered to keep species corresponding to at least 0.5% of annotated reads.…”