title: Universal Human Papillomavirus Typing Assay: Whole-Genome Sequencing following Target Enrichment.
abstract: We designed a universal human papillomavirus (HPV) typing assay based on target enrichment and whole-genome sequencing (eWGS). The RNA bait included 23,941 probes targeting 191 HPV types and 12 probes targeting beta-globin as a control. We used the Agilent SureSelect XT2 protocol for library preparation, Illumina HiSeq 2500 for sequencing, and CLC Genomics Workbench for sequence analysis. Mapping stringency for type assignment was determined based on 8 (6 HPV-positive and 2 HPV-negative) control samples. Using the optimal mapping conditions, types were assigned to 24 blinded samples. eWGS results were 100% concordant with Linear Array (LA) genotyping results for 9 plasmid samples and fully or partially concordant for 9 of the 15 cervical-vaginal samples, with 95.83% overall type-specific concordance for LA genotyping. eWGS identified 7 HPV types not included in the LA genotyping. Since this method does not involve degenerate primers targeting HPV genomic regions, PCR bias in genotype detection is minimized. With further refinements aimed at reducing cost and increasing throughput, this first application of eWGS for universal HPV typing could be a useful method to elucidate HPV epidemiology.
authors.affiliation: Division of High-Consequence Pathogens & Pathology, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
authors.forename: Tengguo
authors.initials: T
authors.lastname: Li
authors.affiliation: Division of High-Consequence Pathogens & Pathology, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
authors.forename: Elizabeth R
authors.initials: ER
authors.lastname: Unger
authors.affiliation: Division of Scientific Resources, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
authors.forename: Dhwani
authors.initials: D
authors.lastname: Batra
authors.affiliation: Division of Scientific Resources, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
authors.forename: Mili
authors.initials: M
authors.lastname: Sheth
authors.affiliation: Division of High-Consequence Pathogens & Pathology, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
authors.forename: Martin
authors.initials: M
authors.lastname: Steinau
authors.affiliation: Agilent Technologies, Inc., Santa Clara, California, USA.
authors.forename: Jean
authors.initials: J
authors.lastname: Jasinski
authors.affiliation: Agilent Technologies, Inc., Santa Clara, California, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Jones
authors.affiliation: Division of High-Consequence Pathogens & Pathology, Centers for Disease Control and Prevention, Atlanta, Georgia, USA mor4@cdc.gov.
authors.forename: Mangalathu S
authors.initials: MS
authors.lastname: Rajeevan
mesh_terms: D016679:Genome, Viral; D023281:Genomics; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D027383:Papillomaviridae / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D030361:Papillomavirus Infections / Q000175:diagnosis / Q000821:virology*; D017422:Sequence Analysis, DNA
keywords: HPV typing; broad-spectrum assay; target enrichment; whole-genome sequencing