Accurate human papillomavirus genotyping by 454 pyrosequencing

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.