Comparative characterization of the mucosal eukaryotic virome across independent whole-metagenome shotgun sequencing cohorts

The human mucosal eukaryotic virome remains incompletely characterized because methodological heterogeneity limits direct comparisons across independent whole-metagenome shotgun sequencing (WMS) datasets. To address this limitation, we reanalyzed publicly available DNA-based WMS datasets representing healthy body-site samples together with nasopharyngeal carcinoma (NPC), human papillomavirus (HPV)-associated disease, and oral squamous cell carcinoma (OSCC) using the same ViromeScan-based analytical approach. Viral count profiles were converted to within-sample relative abundances for Bray–Curtis analyses, host-unmapped read depth was included as a covariate in PERMANOVA, and alpha-diversity findings were evaluated using repeated rarefaction. Anatomical site remained associated with eukaryotic virome diversity and community structure in the HMP cohort after accounting for sequencing depth, and site-associated differences were also observed in the independent NPC cohort analyzed using the same approach. HPV infection status was also associated with viral community structure after depth adjustment, whereas DOI-defined OSCC groups showed no significant global differences in alpha diversity, normalized viral recovery, or community structure and yielded only exploratory taxon-level signals. Across datasets, viral recovery varied with host-unmapped sequencing depth, with substantial between-sample heterogeneity. Together, these findings show that consistent application of publicly available analytical tools can support comparative characterization of eukaryotic virome profiles from existing WMS datasets, while also highlighting the importance of sequencing depth and cautious interpretation of taxon-level associations.