During the recent years, a few studies have described different NGS based approaches to study HPV variability and integration in the human genome. The development of next generation sequencing (NGS) technologies has provided new tools for viral genomic research. Still, several important questions remain for HPV integration and more comprehensive analyses of integration sites are needed in order to expand our understanding of HPV pathogenesis. The finding of certain chromosomal clusters of integration in precancerous lesions and cancers 28 also suggests a selective advantage of specific HPV integrations. Viral integration may also lead to modified expression of cellular genes nearby, disruption of genes, as well as genomic amplifications that may promote oncogenesis 23, 27. Upon integration, disruption or complete deletion of the E1 or E2 gene is often observed, resulting in constitutive expression of the E6 and E7 oncogenes 24, 25, 26, inactivation of cell cycle checkpoints and genetic instability 23. In contrast to a limited number of studies on HPV variability, HPV integration into the host genome has been more widely studied and is regarded as a determining event in cervical carcinogenesis 21, 22, 23. However, recent studies have revealed variability below the level of variant lineages that may be evidence of intra-host viral evolution and adaptation 17, 18, 19, 20. Traditionally, studies have focused on cancer risk of main variants. Isolates of the same HPV types that differ by 1–10% or 0.5–1% across the genome are referred to as variant lineages or sublineages, respectively 11, 12.ĭespite phylogenetic relatedness, HPV variant lineages can differ in their carcinogenic potential 13, 14, 15, 16. Each individual HPV type shares at least 90% sequence identity in the conserved L1 open reading frame (ORF) nucleotide sequence. To date, more than 200 HPV types have been identified 10. HPV contains an approximately 7.9 kb circular double-stranded DNA genome, consisting of early region (E1, E2, E4-7) genes, late region (L1, L2) genes and an upstream regulatory region (URR) 9. An appropriate sequencing approach is needed to uncover these genomic events during a persistent HPV infection. This indicates that in addition to HPV infection, additional factors such as HPV genomic variability and integration, could contribute to the HPV-induced carcinogenic process.
However, only a small fraction of HPV infections at any site will progress to cancer 8. HPV infection is also associated with cancer in penis, vulva, vagina, anus, and head and neck 7.
Of the 13 carcinogenic high-risk types, HPV16 and 18 are associated with about 70% of all cervical cancers 5, 6. A persistent infection with HPV high-risk genotypes is recognised as a necessary cause of cancer development 4. Human papillomavirus (HPV) is the main cause of cervical cancer 1, one of the most common cancers in women worldwide, causing more than 200,000 deaths each year 2, 3. Efficient identification of both HPV variability and integration sites will be important for the study of HPV evolution and adaptability and may be an important tool for use in cervical cancer diagnostics. Comprehensive studies on HPV intra-host variability generated during a persistent infection will improve our understanding of viral carcinogenesis. In contrast to other approaches, TaME-seq proved to be highly efficient in HPV target enrichment, leading to reduced sequencing costs. HPV genomic variability was observed in all samples allowing identification of low frequency variants. Chromosomal integration breakpoints and large deletions were identified in HPV positive cell lines and in one clinical sample. Our results showed deep HPV genome-wide sequencing coverage. For method validation, cell lines (n = 4), plasmids (n = 3), and HPV16, 18, 31, 33 and 45 positive clinical samples (n = 21) were analysed.
TaME-seq combines tagmentation and multiplex PCR enrichment for simultaneous analysis of HPV variation and chromosomal integration, and it can also be adapted to other viruses. To uncover these genomic events in an HPV infection, we have developed an innovative and cost-effective sequencing approach named TaME-seq (tagmentation-assisted multiplex PCR enrichment sequencing). HPV genomic variability and chromosomal integration are important in the HPV-induced carcinogenic process.
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