Optimization of Viral DNA Extraction from Vaginal Swabs

Abstract

The vaginal virome remains under-characterized in healthy individuals and even less so in people with gynecologic conditions, despite its role in the maintenance of microbial homeostasis and potential for clinical application. In conditions such as vulvovaginal candidiasis (VVC), the bacterial composition of the vagina is altered in an attempt to provide defense; however, the bacterial viruses, or bacteriophages (phages), present in this environment have yet to be identified. Thus, this project aimed to establish a protocol for the extraction of viral DNA from vaginal swabs to allow for the further characterization of the vaginal virome. Initially, we applied the established virome extraction protocol using the QIAGEN QIAamp MinElute Virus Spin Kit, optimized for fecal samples. This approach had limited success due to the low biomass swabs used as input. A modified TRIzol protocol with post-purification human DNA depletion using the NEBNext Microbiome DNA Enrichment Kit yielded sequenceable DNA libraries, although with significant human DNA contamination. Our final optimized protocol begins with lysis of host cells prior to virion DNA extraction with the Cytiva Virus Pathogen kit. Our optimized protocol proved to be successful in the extraction of DNA from vaginal swabs, with minimal human DNA contamination and successful enrichment of viral reads in the final dataset. Future work using this protocol will focus on the characterization of bacteriophages present in the vaginal environment and determine how they differ in health and disease, such as in the context of VVC.