title: Deep sequencing of HPV16 genomes: A new high-throughput tool for exploring the carcinogenicity and natural history of HPV16 infection.
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.
authors.affiliation: Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Rockville, MD, USA ; Cancer Genomics Research Laboratory, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Cullen
authors.affiliation: Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Rockville, MD, USA ; Cancer Genomics Research Laboratory, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD, USA.
authors.forename: Joseph F
authors.initials: JF
authors.lastname: Boland
authors.affiliation: Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Rockville, MD, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Schiffman
authors.affiliation: Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Rockville, MD, USA ; Cancer Genomics Research Laboratory, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD, USA.
authors.forename: Xijun
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Rockville, MD, USA.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Wentzensen
authors.affiliation: Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Rockville, MD, USA ; Cancer Genomics Research Laboratory, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD, USA.
authors.forename: Qi
authors.initials: Q
authors.lastname: Yang
authors.affiliation: Department of Epidemiology and Population Health, At Albert Einstein College of Medicine, Bronx, NY, USA.
authors.forename: Zigui
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Rockville, MD, USA.
authors.forename: Kai
authors.initials: K
authors.lastname: Yu
authors.affiliation: Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Rockville, MD, USA ; Cancer Genomics Research Laboratory, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD, USA.
authors.forename: Jason
authors.initials: J
authors.lastname: Mitchell
authors.affiliation: Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Rockville, MD, USA ; Cancer Genomics Research Laboratory, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD, USA.
authors.forename: David
authors.initials: D
authors.lastname: Roberson
authors.affiliation: Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Rockville, MD, USA ; Cancer Genomics Research Laboratory, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD, USA.
authors.forename: Sara
authors.initials: S
authors.lastname: Bass
authors.affiliation: Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Rockville, MD, USA ; Cancer Genomics Research Laboratory, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD, USA.
authors.forename: Laurie
authors.initials: L
authors.lastname: Burdette
authors.affiliation: Department de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.
authors.forename: Moara
authors.initials: M
authors.lastname: Machado
authors.affiliation: Frederick National Laboratory for Cancer Research, Simulation, Analysis and Mathematical Modeling Group Advanced Biomedical Computing Center, Frederick, MD, USA.
authors.forename: Sarangan
authors.initials: S
authors.lastname: Ravichandran
authors.affiliation: Frederick National Laboratory for Cancer Research, Simulation, Analysis and Mathematical Modeling Group Advanced Biomedical Computing Center, Frederick, MD, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Luke
authors.affiliation: Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Rockville, MD, USA.
authors.forename: Mitchell J
authors.initials: MJ
authors.lastname: Machiela
authors.affiliation: Thermo Fisher Scientific (Formerly Life Technologies), Carlsbad, CA, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Andersen
authors.affiliation: Thermo Fisher Scientific (Formerly Life Technologies), Carlsbad, CA, USA.
authors.forename: Matt
authors.initials: M
authors.lastname: Osentoski
authors.affiliation: Thermo Fisher Scientific (Formerly Life Technologies), Carlsbad, CA, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Laptewicz
authors.affiliation: Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Rockville, MD, USA.
authors.forename: Sholom
authors.initials: S
authors.lastname: Wacholder
authors.affiliation: Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Rockville, MD, USA ; Cancer Genomics Research Laboratory, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD, USA.
authors.forename: Ashlie
authors.initials: A
authors.lastname: Feldman
authors.affiliation: Regional Laboratory and Women's Health Research Institute, Division of Research, Kaiser Permanente Northern California, Oakland, CA, USA.
authors.forename: Tina
authors.initials: T
authors.lastname: Raine-Bennett
authors.affiliation: Regional Laboratory and Women's Health Research Institute, Division of Research, Kaiser Permanente Northern California, Oakland, CA, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Lorey
authors.affiliation: Department of Epidemiology and Population Health, At Albert Einstein College of Medicine, Bronx, NY, USA ; Global Coalition Against Cervical Cancer, Arlington, VA, USA.
authors.forename: Philip E
authors.initials: PE
authors.lastname: Castle
authors.affiliation: Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Rockville, MD, USA ; Cancer Genomics Research Laboratory, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD, USA.
authors.forename: Meredith
authors.initials: M
authors.lastname: Yeager
authors.affiliation: Department of Epidemiology and Population Health, At Albert Einstein College of Medicine, Bronx, NY, USA ; Division of Gynecologic Oncology, Department of Obstetrics & Gynecology and Women's Health, at Albert Einstein College of Medicine, Bronx, NY, USA.
authors.forename: Robert D
authors.initials: RD
authors.lastname: Burk
authors.affiliation: Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Rockville, MD, USA.
authors.forename: Lisa
authors.initials: L
authors.lastname: Mirabello
keywords: HPV epidemiology; HPV genomics; HPV16