Nanopore-based shallow shotgun metagenomic sequencing and Illumina-based 16S sequencing of vaginal microbiomes

The vaginal microbiome plays a crucial role in female health, influencing reproductive success, susceptibility to sexually transmitted infections, and the most prevalent vaginal condition in reproductive-age women, bacterial vaginosis (BV). Established approaches for characterizing the vaginal microbiome rely on 16S sequencing, but shallow shotgun metagenomic sequencing (SMS), particularly with Oxford Nanopore technology, offers key advantages in cost-effectiveness, speed, and flexible multiplexing.This study assessed the applicability of Nanopore-based shallow SMS for vaginal microbiome characterization, directly comparing it to Illumina 16S sequencing. The evaluation included 52 women, 23 of whom were diagnosed with BV. A vaginal swab was collected from each participant, and total DNA was extracted, yielding 52 biological samples. Each sample underwent sequencing via both Illumina 16S and Nanopore-based shallow SMS, allowing a direct platform comparison. Reads classified as human (Homo sapiens) by Kraken2 were removed from the shallow SMS data.