Abstract
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.
Highlights
The human papillomaviruses (HPVs) of the genus Alphapapillomavirus are widely prevalent among human populations, infecting anogenital and oral mucosal and cutaneous epithelia [1]
We whole-genome sequenced 796 HPV type 16 (HPV16)-positive specimens and report several metrics to demonstrate the high quality and reliability of our assay and describe HPV16 genome variation based on these next-generation sequencing (NGS) data
We developed and implemented a high-throughput, NGS technique based on Ampliseq and Ion Torrent technology to investigate the nucleotide changes associated with human papillomavirus oncogenicity
Summary
The human papillomaviruses (HPVs) of the genus Alphapapillomavirus are widely prevalent among human populations, infecting anogenital and oral mucosal and cutaneous epithelia [1]. Persistent infection with one of a dozen carcinogenic HPV types is a well-established, necessary cause of cervical cancer [3], the third leading cause of cancer in women worldwide [4]. Despite the pervasiveness of “high-risk” HPV (HR-HPV) infections, only a small fraction of women with a HR-HPV infection at any infected site will progress to precancer or cancer [5,6]. This indicates that additional risk factors are important for HR-HPV carcinogenesis, potentially including viral genetic factors; these factors remain poorly understood
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