Cervicovaginal microbiome and HPV: A standardized workflow from 16S/ITS NGS to microbial community profiling

16S rRNA next-generation sequencing (NGS) has significantly advanced cervicovaginal microbiome profiling, offering insights into the relationship between vaginal dysbiosis and HPV-associated carcinogenesis. However, reliance on the 16S V3V4 hypervariable region alone introduces inherent biases that impact results. This study developed standardized workflows for 16S-ITS NGS, with a focus on identifying methodological biases that influence microbial abundance and taxonomic specificity. Commercial NGS tools were employed, including the 16S-ITS QIAseq V1-V9 screening panel, ATCC vaginal microbial standard, and CLC Genomics Workbench integrated with a customized database (VAGIBIOTA) for analysis. The microbial communities of 66 cervical cytology samples were characterized. Among the regions tested, V3V4 exhibited the least quantitative bias, while V1V2 offered the highest specificity. Microbial profiles and Community State Types (CSTs) (I-V) were consistent with prior studies, with Lactobacillus abundance clustering into three states: L.-dominant (CST I-III, V), L-diminished (CST IV-A), and L.-depleted (CST IV-B). Differential abundance analysis revealed that anaerobic opportunistic pathogens predominant in CST IV-B (dysbiosis) were also enriched in HSIL and HPV-16 positive samples. Our findings highlight the importance of standardizing workflows by incorporating the full-spectrum V1-V9 panel for microbial community profiling and elucidating their contributions to HPV-associated carcinogenesis.