A Cost Effective, Rapid, and Portable Approach to Characterize HPV L1 Genomic Variability for Population Screening Using Nanopore Sequencing
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Human papillomavirus (HPV) is associated with cervical cancer, as also cancers of the head and neck, penis, vulva, vagina, and anus. Per the GLOBOCAN 2018 [1] estimates of cancer incidence and mortality—there were 570000 cases, and 311000 deaths due to cervical cancer worldwide. Similarly, 600000 cases of head and neck cancer (HNC) were reported in 2018. HPV is linked to a subset of head and neck squamous cell carcinoma (HNSCC), particularly oropharyngeal cancer. Not all HPV infections progress to cancer. Persistent infection with HPV high-risk genotypes; and genomic variability and integration are critical in HPV induced carcinogenesis. We have developed a cost-effective approach for characterisation of HPV L1 genotypes and variability using Nanopore sequencing. The assay consists of amplification of 450 base HPV L1 region by PCR using degenerate HPV primers followed by sequencing of positive samples using long read Nanopore sequencing. This is followed by analysis of sequence variants and deriving a risk score based on the variants. For bench marking and validation of the method, we used cell lines (n=3), and positive clinical samples in HPV16, 18, 31, 33 and 45. Portability of the sequencer, flexibility of scale, and low reagent cost of the assay [21 USD per sample] make Nanopore sequencing a valuable tool for large scale HPV screening programs in high risk populations.