title: Deep sequencing of HPV E6/E7 genes reveals loss of genotypic diversity and gain of clonal dominance in high-grade intraepithelial lesions of the cervix.
abstract: Human papillomavirus (HPV) is the carcinogen of almost all invasive cervical cancer and a major cause of oral and other anogenital malignancies. HPV genotyping by dideoxy (Sanger) sequencing is currently the reference method of choice for clinical diagnostics. However, for samples with multiple HPV infections, genotype identification is singular and occasionally imprecise or indeterminable due to overlapping chromatograms. Our aim was to explore and compare HPV metagenomes in abnormal cervical cytology by deep sequencing for correlation with disease states.
  
  
  
  Low- and high-grade intraepithelial lesion (LSIL and HSIL) cytology samples were DNA extracted for PCR-amplification of the HPV E6/E7 genes. HPV+ samples were sequenced by dideoxy and deep methods. Deep sequencing revealed ~60% of all samples (n = 72) were multi-HPV infected. Among LSIL samples (n = 43), 27 different genotypes were found. The 3 dominant (most abundant) genotypes were: HPV-39, 11/43 (26%); -16, 9/43 (21%); and -35, 4/43 (9%). Among HSIL (n = 29), 17 HPV genotypes were identified; the 3 dominant genotypes were: HPV-16, 21/29 (72%); -35, 4/29 (14%); and -39, 3/29 (10%). Phylogenetically, type-specific E6/E7 genetic distances correlated with carcinogenic potential. Species diversity analysis between LSIL and HSIL revealed loss of HPV diversity and domination by HPV-16 in HSIL samples.
  
  
  
  Deep sequencing resolves HPV genotype composition within multi-infected cervical cytology. Biodiversity analysis reveals loss of diversity and gain of dominance by carcinogenic genotypes in high-grade cytology. Metagenomic profiles may therefore serve as a biomarker of disease severity and a population surveillance tool for emerging genotypes.
authors.affiliation: Department of Clinical Investigation, Brooke Army Medical Center, Gynecologic Oncology & Clinical Investigation, 3698 Chambers Pass, Fort Sam Houston, TX, 78234, USA. jane.shengunther.mil@mail.mil.
authors.forename: Jane
authors.identifier: 0000-0002-0876-9338
authors.initials: J
authors.lastname: Shen-Gunther
authors.affiliation: Department of Biology, University of Texas at San Antonio, San Antonio, TX, 78249, USA.
authors.forename: Yufeng
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Greehey Children's Cancer Research Institute, University of Texas Health Science Center at San Antonio, San Antonio, TX, 78229, USA.
authors.forename: Zhao
authors.initials: Z
authors.lastname: Lai
authors.affiliation: Department of Clinical Investigation, Brooke Army Medical Center, Gynecologic Oncology & Clinical Investigation, 3698 Chambers Pass, Fort Sam Houston, TX, 78234, USA.
authors.forename: Graham M
authors.initials: GM
authors.lastname: Poage
authors.affiliation: Department of Clinical Investigation, Brooke Army Medical Center, Gynecologic Oncology & Clinical Investigation, 3698 Chambers Pass, Fort Sam Houston, TX, 78234, USA.
authors.forename: Luis
authors.initials: L
authors.lastname: Perez
authors.affiliation: Department of Molecular Medicine, Cancer Therapy and Research Center, University of Texas Health Science Center at San Antonio, San Antonio, TX, 78229, USA.
authors.forename: Tim H M
authors.initials: TH
authors.lastname: Huang
mesh_terms: D001483:Base Sequence; D004279:DNA, Viral / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019143:Evolution, Molecular; D005260:Female; D056426:Genetic Loci; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D060787:Neoplasm Grading; D009856:Oncogene Proteins, Viral / Q000145:classification / Q000235:genetics*; D027383:Papillomaviridae / Q000235:genetics* / Q000378:metabolism; D050725:Papillomavirus E7 Proteins / Q000145:classification / Q000235:genetics*; D010802:Phylogeny; D017422:Sequence Analysis, DNA; D002583:Uterine Cervical Neoplasms / Q000473:pathology / Q000821:virology
chemical_list: D004279:DNA, Viral; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins
keywords: Deep sequencing; HPV genotyping; HSIL; High-throughput sequencing; Human papillomavirus; LSIL; Metagenome; Virome