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

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          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. The PVAmpliconFinder workflow, along with its source code, is freely available on the GitHub platform: https://github.com/IARCbioinfo/PVAmpliconFinder.

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          Most cited references22

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          The virome in mammalian physiology and disease.

          The virome contains the most abundant and fastest mutating genetic elements on Earth. The mammalian virome is constituted of viruses that infect host cells, virus-derived elements in our chromosomes, and viruses that infect the broad array of other types of organisms that inhabit us. Virome interactions with the host cannot be encompassed by a monotheistic view of viruses as pathogens. Instead, the genetic and transcriptional identity of mammals is defined in part by our coevolved virome, a concept with profound implications for understanding health and disease. Copyright © 2014 Elsevier Inc. All rights reserved.
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            Performance, Accuracy, and Web Server for Evolutionary Placement of Short Sequence Reads under Maximum Likelihood

            We present an evolutionary placement algorithm (EPA) and a Web server for the rapid assignment of sequence fragments (short reads) to edges of a given phylogenetic tree under the maximum-likelihood model. The accuracy of the algorithm is evaluated on several real-world data sets and compared with placement by pair-wise sequence comparison, using edit distances and BLAST. We introduce a slow and accurate as well as a fast and less accurate placement algorithm. For the slow algorithm, we develop additional heuristic techniques that yield almost the same run times as the fast version with only a small loss of accuracy. When those additional heuristics are employed, the run time of the more accurate algorithm is comparable with that of a simple BLAST search for data sets with a high number of short query sequences. Moreover, the accuracy of the EPA is significantly higher, in particular when the sample of taxa in the reference topology is sparse or inadequate. Our algorithm, which has been integrated into RAxML, therefore provides an equally fast but more accurate alternative to BLAST for tree-based inference of the evolutionary origin and composition of short sequence reads. We are also actively developing a Web server that offers a freely available service for computing read placements on trees using the EPA.
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              A broad range of human papillomavirus types detected with a general PCR method suitable for analysis of cutaneous tumours and normal skin.

              A pair of degenerate PCR primers (FAP59/64) was designed from two relatively conserved regions of the L1 open reading frame of most human papillomaviruses (HPV). The size of the generated amplicon was about 480 bp. PCR using these primers was found capable of amplifying DNA from 87% (65/75) of the HPV types tested, its sensitivity being 1-10 copies for HPV-5, -20 and -30 clones. HPV was found in 63% (5/8) of tumour samples and in 63% (5/8) of normal skin biopsies from patients with various cutaneous tumours. HPV-5, HPV-8, HPV-12, HPVvs20-4 and six putatively novel HPV types were identified. No correlation was found to exist between specific HPV and tumour types. Skin surface swab samples from one or more sites on three of four healthy volunteers were found to contain HPV, types 12 and 49 being identified, as well as eight novel HPV types, two of which were also found among the patients. In all, HPV was detected in 75% (9/12) of those tested, five HPV types and 12 novel candidate types being identified, and 37% (7/19) of HPV-positive samples were found to manifest more than one HPV type. All the HPV detected manifested high degrees of nucleotide sequence similarity with HPV types associated with skin lesions and epidermodysplasia verruciformis. The overall HPV finding in the skin samples was 50% (20/40) using the FAP primers as compared to 18% (7/40) using another PCR test designed for skin types. The results thus suggest the new method to be sensitive and generally applicable for detecting cutaneous HPV.
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                Author and article information

                Contributors
                alexis.robitaille@orange.fr
                tommasinom@iarc.fr
                olivierm@iarc.fr
                Journal
                BMC Bioinformatics
                BMC Bioinformatics
                BMC Bioinformatics
                BioMed Central (London )
                1471-2105
                8 June 2020
                8 June 2020
                2020
                : 21
                : 233
                Affiliations
                [1 ]GRID grid.17703.32, ISNI 0000000405980095, International Agency for Research on Cancer, ; Lyon, France
                [2 ]GRID grid.468198.a, ISNI 0000 0000 9891 5233, Department of Cancer Epidemiology, , Moffitt Cancer Center, ; Tampa, Florida USA
                [3 ]GRID grid.9845.0, ISNI 0000 0001 0775 3222, Institute of Clinical and Preventive Medicine, , University of Latvia, ; Riga, Latvia
                [4 ]GRID grid.452463.2, German Center for Infection Research, , Hamburg-Borstel-Lübeck-Riems, ; Hamburg, Germany
                [5 ]GRID grid.13648.38, ISNI 0000 0001 2180 3484, Institute for Medical Microbiology, Virology and Hygiene, , University Medical Center Hamburg-Eppendorf, ; Hamburg, Germany
                [6 ]GRID grid.418481.0, ISNI 0000 0001 0665 103X, Heinrich Pette Institut, , Leibniz Institut for Experimental Virology, ; Hamburg, Germany
                [7 ]GRID grid.418573.c, Department of Oncogene Regulation, , Chittaranjan National Cancer Institute, ; Kolkata, India
                Author information
                http://orcid.org/0000-0002-9749-8276
                Article
                3573
                10.1186/s12859-020-03573-8
                7282039
                32513098
                f3cc9d64-48e4-4bfb-9658-ac76d9b81c34
                © The Author(s) 2020

                Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

                History
                : 28 October 2019
                : 28 May 2020
                Funding
                Funded by: Institut National de la Santé et de la Recherche Médicale
                Award ID: ENV201610
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100004097, Fondation ARC pour la Recherche sur le Cancer;
                Award ID: JA 20151203192
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100005972, Deutsche Krebshilfe;
                Award ID: no. 110259
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000054, National Cancer Institute;
                Award ID: 1R01-CA17758
                Award Recipient :
                Categories
                Methodology Article
                Custom metadata
                © The Author(s) 2020

                Bioinformatics & Computational biology
                amplicon sequencing,virus discovery,papillomavirus,workflow,phylogeny

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