PVAmpliconFinder: a workflow for the identification of human papillomaviruses from high-throughput amplicon sequencing

Abstract

Background: The detection of known human papillomaviruses (PVs) from targeted wet-lab approaches has traditionally used PCR-based methods coupled with Sanger sequencing. With the introduction of next-generation sequencing (NGS), these approaches can be revisited to integrate the sequencing power of NGS. Although computational tools have been developed for metagenomic approaches to search for known or novel viruses in NGS data, no appropriate tool is available for the classification and identification of novel viral sequences from data produced by amplicon-based methods. Results: We have developed PVAmpliconFinder, a data analysis workflow designed to rapidly identify and classify known and potentially new Papillomaviridae sequences from NGS amplicon sequencing with degenerate PV primers. Here, we describe the features of PVAmpliconFinder and its implementation using biological data obtained from amplicon sequencing of human skin swab specimens and oral rinses from healthy individuals. Conclusions: PVAmpliconFinder identified putative new HPV sequences, including one that was validated by wet-lab experiments. PVAmpliconFinder can be easily modified and applied to other viral families. PVAmpliconFinder addresses a gap by providing a solution for the analysis of NGS amplicon sequencing, increasingly used in clinical research.

Diversity of human papillomavirus in the anal canal of HIV-positive and HIV-negative men

“…However, the classification of these PVs as animal types could be due to the limited information on the HPV L1 sequence, and the consideration of additional parts of the genome could result in a different classification as closely related HPVs. 19 In conclusion, using a well-validated NGS strategy, this study expanded our knowledge on HPV diversity in the anal mucosa. It showed that, besides infections with alpha HPVs, many beta and gamma HPV types from different species colonize the anal mucosal epithelium.…”

“…17 , 18 Bioinformatic analysis was performed using the bioinformatic pipeline PVAmpliconFinder. 19 All the results in this study were based on the identification of the sequences following the homology-based classification using the evolutionary placement algorithm (EPA) in RAxML (Randomized Axelerated Maximum Likelihood). Only the longest sequence was considered for RAxML-EPA classification when several singlets or contigs were clustered together by PVAmpliconFinder.…”

Evaluation of human papillomavirus DNA in colorectal cancer and adjacent mucosal tissue samples

“…Alternatively, this result can also be explained by a lack of specificity of the amplification due to the use of degenerated primers. We also detected DNA sequences from several beta HPV types from species beta-1, beta-2 and beta-3 (HPV20, 23,24,49,93,98,124) and from gamma-1 (HPV205) (Tables 3, 4). Cutaneous beta HPV types, specifically beta-2 HPV111 (n = 3) and HPV120 (n = 1), were only detected by Luminex assay in DNA extracted from surrounding tissues.…”

“…NGS analysis was performed on the pooled amplicon-based library (Nextera DNA Flex) using the Illumina MiSeq sequencer (2 X 150 paired-end reads, MiSeq reagent kit v3) (Illumina, San Diego, CA, USA) as previously reported [21]. Bioinformatic analyses were performed using PVAmpliconFinder tool [23], and all the results were based on the homology-based classification using the evolutionary placement algorithm in RAxML (Randomized Axelerated Maximum Likelihood) [24].…”