Next generation sequencing for human papillomavirus genotyping

Abstract

Background: Human papillomavirus (HPV) genotyping using next generation sequencing (NGS) could be useful to study the HPV variant-specific epidemiology, including monitoring for possible emergence of new HPV variants after introduction of HPV vaccination programs.

Objectives: We wished to design and validate a method for rapid HPV detection, typing and sequencing in clinical samples.

Study design: Plasmids of 15 different HPV types were mixed and serially diluted in human DNA in concentrations ranging from 1 to 100 copies per sample, amplified using the HPV general PCR primer pair PGMY and sequenced using 454 technology. Sixty cervical samples were tested both with the NGS-based method and with a comparison method based on genotyping using type-specific probes bound to fluorescent beads (Luminex). Thirty-three clinical samples were repeat tested using NGS to evaluate reproducibility.

Results: The NGS-based method correctly identified all 15 mixed HPV types when present in 100 copies/sample and 13/15 types when present in 10 copies/sample. For 36/60 cervical samples genotyping results using NGS and Luminex were identical. For 12/60 samples the NGS method was more sensitive than the Luminex test and most of the remaining discrepancies could be explained by the different type coverage of the assays. Reproducibility testing found complete or partial concordance in 30/33 samples.

Conclusions: NGS provides a sensitive and accurate method for genotyping of HPV. The fact that also the amplimer sequence is obtained could be important for studying the epidemiology of viral variants and monitoring of HPV vaccination.

Keywords: CIN; GS; GS junior; HPV; High throughput sequencing; Human papillomavirus; Luminex; MID; NGS; Next generation sequencing; cervical intraepithelial neoplasia; emPCR; emulsion PCR; genome sequencer; human papillomavirus; multiplex identifier; next generation sequencing.