Revisiting HPV integration landscapes using telomere-to-telomere human genome reference closes the gaps in HPV integration sites search.

Accurate characterization of human papillomavirus (HPV) integration into the host genome is essential for understanding HPV-driven carcinogenesis. However, most previous integration studies relied on incomplete human reference genomes, potentially obscuring integration events occurring within highly repetitive genomic regions. Here, we reassessed HPV integration landscapes using the first complete telomere-to-telomere (T2T) human reference genome assembly (hs1). We reanalyzed datasets from three independent studies generated using complementary sequencing technologies, including short-read Illumina sequencing, PacBio continuous long reads (CLR), PacBio high-fidelity (HiFi) sequencing, and ultra-long Oxford Nanopore sequencing. Reanalysis using the T2T reference confirmed the majority of previously reported integration breakpoints while identifying numerous additional sites, increasing the total number of detected breakpoints from 446 to 799. Notably, newly detected integrations frequently localized to repeat-rich genomic regions, including centromeric and pericentromeric satellite sequences that were absent from earlier reference assemblies. Importantly, integration events within these regions were detectable not only in long-read datasets but also in short-read sequencing data when analyzed using the complete reference genome. Our findings demonstrate that incomplete reference assemblies have led to systematic underestimation of HPV integration events, particularly within structurally complex genomic regions. These results highlight the importance of adopting complete human genome references for viral integration analyses and suggest that reanalysis of existing datasets may reveal previously overlooked biologically and clinically relevant integration events.