title: Accurate human papillomavirus genotyping by 454 pyrosequencing.
abstract: Accurate HPV typing is essential for evaluation and monitoring of HPV vaccines, for second-line testing in cervical cancer screening, and in epidemiological surveys. In this study, we set up and assessed in clinical samples a new HPV typing method based on 454 next-generation sequencing (NGS) of HPV L1 amplicons, generated by using a modified PGMY primer set with improved sensitivity for some HPV types that are not targeted by standard PGMY primers. By using a median 12 800-fold coverage, the NGS method allowed us to correctly identify all high-risk HPV types, in either single or multiple infections, with a sensitivity of 50 genome equivalents, as demonstrated by testing WHO LabNet EQA sample panels. Analysis of mixtures of HPV16- and HPV18-positive cell lines demonstrated that the NGS method could reproducibly quantify the proportion of each HPV type in multiple infections in a wide dynamic range. Testing of HPV-positive clinical samples showed that NGS could correctly identify a high number of HPV types in multiple infections. The NGS method was also effective in the analysis of a set of cervical specimens with discordant results at hybrid capture 2 and line probe assays. In conclusion, a new HPV typing method based on 454 pyrosequencing was set up. This method was sensitive, specific, quantitative and precise in both single and multiple infections. It could identify a wide range of HPV types and might potentially discover new HPV types.
authors.affiliation: Department of Molecular Medicine, University of Padova, Padova, Italy.
authors.forename: V
authors.initials: V
authors.lastname: Militello
authors.forename: E
authors.initials: E
authors.lastname: Lavezzo
authors.forename: G
authors.initials: G
authors.lastname: Costanzi
authors.forename: E
authors.initials: E
authors.lastname: Franchin
authors.forename: B
authors.initials: B
authors.lastname: Di Camillo
authors.forename: S
authors.initials: S
authors.lastname: Toppo
authors.forename: G
authors.initials: G
authors.lastname: Palù
authors.forename: L
authors.initials: L
authors.lastname: Barzon
mesh_terms: D005835:Genitalia / Q000821:virology; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D058889:Molecular Typing / Q000379:methods*; D027383:Papillomaviridae / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D030361:Papillomavirus Infections / Q000175:diagnosis / Q000821:virology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
keywords: 454 pyrosequencing; diagnostic accuracy; genotyping; human papillomavirus; multiple infection; next generation sequencing; sensitivity and specificity