For unknown reasons, there is huge variability in risk conferred by different HPV types and, remarkably, strong differences even between closely related variant lineages within each type. HPV16 is a uniquely powerful carcinogenic type, causing approximately half of cervical cancer and most other HPV-related cancers. To permit the large-scale study of HPV genome variability and precancer/cancer, starting with HPV16 and cervical cancer, we developed a high-throughput next-generation sequencing (NGS) whole-genome method. We designed a custom HPV16 AmpliSeq?panel that generated 47 overlapping amplicons covering 99% of the genome sequenced on the Ion Torrent Proton platform. After validating with Sanger, the current "gold standard" of sequencing, in 89 specimens with concordance of 99.9%, we used our NGS method and custom annotation pipeline to sequence 796 HPV16-positive exfoliated cervical cell specimens. The median completion rate per sample was 98.0%. Our method enabled us to discover novel SNPs, large contiguous deletions suggestive of viral integration (OR of 27.3, 95% CI 3.3-222, P=0.002), and the sensitive detection of variant lineage coinfections. This method represents an innovative high-throughput, ultra-deep coverage technique for HPV genomic sequencing, which, in turn, enables the investigation of the role of genetic variation in HPV epidemiology and carcinogenesis.
Deep sequencing of HPV16 genomes: A new high-throughput tool for exploring the carcinogenicity and natural history of HPV16 infection
Authors: Cullen M; Boland JF; Schiffman M; Zhang X; Wentzensen N; Yang Q; Chen Z; Yu K; Mitchell J; Roberson D; Bass S; Burdette L; Machado M; Ravichandran S; Luke B; Machiela MJ; Andersen M; Osentoski M; Laptewicz M; Wacholder S; Feldman A; Raine-Bennett T; Lorey T; Castle PE; Yeager M; Burk RD; Mirabello L
Papillomavirus Res. 2015 Dec 1;1:3-11.