Nested PCR followed by NGS: Validation and application for HPV genotyping of Tunisian cervical samples

Abstract

The most used methodologies for HPV genotyping in Tunisian studies are based on hybridization that are limited to a restricted number of HPV types and to a lack of specificity and sensitivity for same types. Recently, Next-Generation sequencing (NGS) technology has been efficiently used for HPV genotyping. In this work we designed and validated a sensitive genotyping method based on nested PCR followed by NGS. Eighty-six samples were tested for the validation of an HPV genotyping assay based on Nested-PCR followed by NGS. These include, 43 references plasmids and 43 positive HPV clinical cervical specimens previously evaluated with the conventional genotyping method: Reverse Line Hybridization (RLH). Results of genotyping using NGS were compared to those of RLH. The analytical sensitivity of the NGS assay was 1GE/μl per sample. The NGS allowed the detection of all HPV types presented in references plasmids. On the clinical samples, a total of 19 HPV types were detected versus 14 types using RLH. Besides the identification of more HPV types in multiple infection (6 types for NGS versus 4 for RLH), NGS allowed the identification of HPV types that were not detected by RLH. In addition, the NGS assay detected newly HPV types that were not described in Tunisia so far: HPV81, HPV43, HPV74, and HPV62. The high sensitivity and specificity of NGS for HPV genotyping in addition to the identification of new HPV types may justify the use of such technique to provide with high accuracy the profile of circulating types in epidemiological studies.

Fig 1. Workflow for library preparation and sequencing.

Amplicons generated through a PGMY PCR were amplified using customized primers containing Miseq specific adaptors then indexation and pooling were done before sequencing using Miseq plateform.

Fig 2. Workflow for data analysis.

The designed pipeline for HPV genotyping in this study contains 3 steps: reads quality control to remove bad quality reads, processing of retained reads for mapping into Human genome, and finally in the final set of selected sequences the viruses were detected and genotyped by assembly and contigs mapping to HPV reference genome.

Fig 3. HPV-aligned reads vs. total number of reads according to HPV status and number of HPV types in the samples.

There was no difference in total reads number between positive samples (blue dots) and negative samples (red dots). In contrast, alignment of HPV reads depended on the HPV types in each positive sample (p<10⁻³): The aligned HPV reads were more numerous in samples having multiple infections (HPV types number >1): the size of dots indicates the number of HPV types in each types sample.

Fig 4. Detected HPV types by NGS and comparison with RLH.