Sampling Considerations and Advantages of Shotgun Metagenomics in Vaginal Microbiome Research

Abstract

The vaginal microbiome comprises a complex microbial ecosystem that plays a crucial role in female reproductive health and has been associated with adverse pregnancy outcomes, infertility, and other gyne¬cological conditions. To date, most vaginal microbiome studies have relied on full-length 16S rRNA gene sequencing, which is limited in taxonomic resolution and often cannot reliably distinguish closely related species. In contrast, shotgun metagenomic sequencing enables species- and strain-level characterization while simultaneously providing comprehensive taxonomic and functional profiling of bacteria, fungi, DNA viruses, and other microorganisms without amplification bias. Despite these advantages, the successful application of shotgun metagenomics to vaginal samples remains technically challenging because of the high proportion of host DNA and relatively low microbial biomass. Methodological variability introduced during sample collection, transport, storage, DNA extraction, host DNA depletion, sequencing, and bio¬informatic analysis can substantially influence taxonomic profiles and compromise the comparability and reproducibility of results. Although numerous studies have demonstrated the analytical advantages of shot¬gun metagenomics, standardized protocols for pre-analytical sample handling and workflow optimization remain limited. This narrative review summarizes current evidence on sampling strategies and meth¬odological factors influencing shotgun metagenomic analysis of the vaginal microbiome. Particular empha¬sis is placed on potential sources of bias throughout the pre-analytical and analytical workflow and their impact on downstream taxonomic profiling. By highlighting critical methodological considerations, this review aims to support improved standardization and enhance the reproducibility, comparability, and clin¬ical translation of vaginal shotgun metagenomic studies.