# Toward accurate vaginal microbiome profiling: protocol, bioinformatics, and core microbiota characterisation

## TL;DR

This study examines the vaginal microbiome in healthy women using sequencing and bioinformatics to identify common microbial patterns and improve profiling accuracy.

## Contribution

The study introduces optimized PCR and bioinformatic methods for nanopore sequencing to better characterize the vaginal microbiome.

## Key findings

- Lactobacillus crispatus dominance (CST I) was the most common vaginal microbiome type in healthy women.
- Nanopore sequencing with Porechop and NanoCLUST provided the most accurate microbial identification.
- C. trachomatis was not detected in any samples despite optimized PCR methods.

## Abstract

Rising demand for assisted reproductive technologies (ART) with limited improvements in success rates has driven interest in the impact of the vaginal microbiome on fertility outcomes. In order to fully examine the relationship between the vaginal microbiome and fertility outcomes, methodologies and technological developments must be standardised and benchmarked to provide the most accurate assessment of microbial population representation.

This study sought to investigate the utility of 16S sequencing and bioinformatic approaches using nanopore sequencing to characterize core vaginal microbiota in a healthy Australian cohort of reproductive-age women.

Optimisation and comparison of different PCR strategies for whole 16S amplification was undertaken, along with the generation of bioinformatic analysis strategies. Initial qPCR identified the 27F-YM (MIX) primer as the most sensitive for C. trachomatis. However, nanopore sequencing revealed no detectable C. trachomatis across all six samples. Among the bioinformatic tools, Porechop with NanoCLUST most accurately identified microbial presence. Community state type (CST) I—characterised by Lactobacillus crispatus dominance—was identified as the most common CST (66%), aligning with patterns of a healthy vaginal microbiome.

The findings highlight a Lactobacillus-rich microbiome as the most common among healthy females; however, further refinement—potentially through a metagenomics approach—is recommended to address 16S rRNA primer limitations to enable improved accuracy of microbial detection for the vaginal microbiome.

The online version contains supplementary material available at 10.1007/s10815-025-03509-2.