title: Targeted full-genome amplification and sequencing of dengue virus types 1-4 from South America.
abstract: We report optimized workflows for full-genome sequencing of dengue viruses (DENVs) 1-4. Based on alignments of publicly available complete genomes we modified and expanded existing primers sets to amplify DENV genotypes that were previously difficult or impossible to sequence. We also report improvements to streamline laboratory handling, including a dual amplification strategy for easy and difficult to sequence "high-copy" and "low-copy" templates, respectively, and normalization of PCR cycling conditions across serotypes. High-copy templates can be sequenced following amplification of as few as 5 overlapping segments covering the complete viral genome, whereas low-copy templates can be sequenced following amplification of no more than 10 overlapping segments of smaller size. These changes have been validated using a balanced set of wild-type DENV genomes (11 of DENV1, 14 of DENV2, 13 of DENV3 and 7 of DENV4) derived from human serum samples collected throughout South America over the past 15 years. The changes described enable generation of complete DENV genomes from wild-type samples without the need for viral enrichment via passaging through laboratory cell lines. This should facilitate quick and cost-effective generation of DENV full-genome sequences of the type needed for accurate epidemiological surveillance and thorough evolutionary studies of wild-type DENVs.
authors.affiliation: U.S. Naval Medical Research Unit No. 6 (NAMRU-6), Av. Venezuela c36s/n, Bellavista, Callao 2, Peru. Electronic address: cristopher.d.cruz.ln@mail.mil.
authors.forename: Cristopher D
authors.initials: CD
authors.lastname: Cruz
authors.affiliation: U.S. Naval Medical Research Unit No. 6 (NAMRU-6), Av. Venezuela c36s/n, Bellavista, Callao 2, Peru. Electronic address: armando.torre@med.navy.mil.
authors.forename: Armando
authors.initials: A
authors.lastname: Torre
authors.affiliation: U.S. Naval Medical Research Unit No. 6 (NAMRU-6), Av. Venezuela c36s/n, Bellavista, Callao 2, Peru. Electronic address: gilda.troncos.fn@mail.mil.
authors.forename: Gilda
authors.initials: G
authors.lastname: Troncos
authors.affiliation: Insect-Virus Interactions Group, Department of Genomes and Genetics, Institut Pasteur, Paris, France; Centre National de la Recherche Scientifique, Unité de Recherche Associée 3012, Paris, France. Electronic address: louis.lambrechts@pasteur.fr.
authors.forename: Louis
authors.initials: L
authors.lastname: Lambrechts
authors.affiliation: U.S. Naval Medical Research Unit No. 6 (NAMRU-6), Av. Venezuela c36s/n, Bellavista, Callao 2, Peru. Electronic address: mariana.leguia.fn@mail.mil.
authors.forename: Mariana
authors.initials: M
authors.lastname: Leguia
mesh_terms: D017931:DNA Primers; D003715:Dengue / Q000821:virology; D003716:Dengue Virus / Q000235:genetics* / Q000302:isolation & purification; D019143:Evolution, Molecular; D016679:Genome, Viral; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D017931:DNA Primers; D012367:RNA, Viral
keywords: Amplicon sequencing; Dengue virus; Full-genome sequencing; Next-generation sequencing