title: Rapid next-generation sequencing of dengue, EV-A71 and RSV-A viruses.
abstract: Accurate characterisation of viral strains constitutes a crucial objective for the management of modern virus collections. Next-generation sequencing (NGS) provides technical solution for fast and cost-effective full genome sequencing. Here, we report protocols for rapid full-genome characterisation of RNA viruses of medical importance: dengue virus, enterovirus A71 and respiratory syncytial virus A, based on a specific amplification step followed by NGS-sequencing. A subset of full-length genome sequences representing the genetic diversity of each virus type was selected in GenBank and used to design primer sets allowing the amplification of the complete genome in 3-8 overlapping PCR fragments. The technique was used for characterising 53 strains (33 DENV, 8 EV-A71, 12 RSV-A) from various genotypes and origins. In a single assay, and in just 4 days, it provided for all strains an excellent genomic coverage (∼ 99% including complete ORF for all strains) and accurate sequences with high number of reads per position (250-3500 on average). The elaboration of specific PCR-based full-genome sequencing protocols for diverse virus groups is likely to revolutionise the characterisation of viral isolates in modern collection, but also to contribute in the next future to the study of RNA viruses directly from biological samples.
authors.affiliation: Aix Marseille Université, IRD French Institute of Research for Development, EHESP French School of Public Health, EPV UMR_D 190 "Emergence des Pathologies Virales", 13005 Marseille, France; IHU Méditerranée Infection, APHM Public Hospitals of Marseille, 13005 Marseille, France. Electronic address: Cecile.baronti@univ-amu.fr.
authors.forename: Cécile
authors.initials: C
authors.lastname: Baronti
authors.affiliation: Aix Marseille Université, IRD French Institute of Research for Development, EHESP French School of Public Health, EPV UMR_D 190 "Emergence des Pathologies Virales", 13005 Marseille, France; IHU Méditerranée Infection, APHM Public Hospitals of Marseille, 13005 Marseille, France.
authors.forename: Géraldine
authors.initials: G
authors.lastname: Piorkowski
authors.affiliation: Aix Marseille Université, IRD French Institute of Research for Development, EHESP French School of Public Health, EPV UMR_D 190 "Emergence des Pathologies Virales", 13005 Marseille, France; French National Reference Centre for Arboviruses, IRBA, 13013 Marseille, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Leparc-Goffart
authors.affiliation: Aix Marseille Université, IRD French Institute of Research for Development, EHESP French School of Public Health, EPV UMR_D 190 "Emergence des Pathologies Virales", 13005 Marseille, France; IHU Méditerranée Infection, APHM Public Hospitals of Marseille, 13005 Marseille, France.
authors.forename: Xavier
authors.initials: X
authors.lastname: de Lamballerie
authors.affiliation: Aix Marseille Université, IRD French Institute of Research for Development, EHESP French School of Public Health, EPV UMR_D 190 "Emergence des Pathologies Virales", 13005 Marseille, France; Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU), Microbiology Laboratory, Mahosot Hospital, PDR Vientiane, Lao Democratic People's Republic; Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Churchill Hospital, Oxford OX3 7LJ, UK.
authors.forename: Audrey
authors.initials: A
authors.lastname: Dubot-Pérès
mesh_terms: D003716:Dengue Virus / Q000235:genetics*; D029821:Enterovirus A, Human / Q000235:genetics*; D016679:Genome, Viral; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics; D012136:Respiratory Syncytial Viruses / Q000235:genetics*; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: D012367:RNA, Viral
keywords: Dengue virus; Enterovirus A71; Human respiratory syncytial virus A; Ion torrent; Next-generation sequencing; Specific amplification