title: Near-complete genome sequencing of swine vesicular disease virus using the Roche GS FLX sequencing platform.
abstract: Swine vesicular disease virus (SVDV) is an enterovirus that is both genetically and antigenically closely related to human coxsackievirus B5 within the Picornaviridae family. SVDV is the causative agent of a highly contagious (though rarely fatal) vesicular disease in pigs. We report a rapid method that is suitable for sequencing the complete protein-encoding sequences of SVDV isolates in which the RNA is relatively intact. The approach couples a single PCR amplification reaction, using only a single PCR primer set to amplify the near-complete SVDV genome, with deep-sequencing using a small fraction of the capacity of a Roche GS FLX sequencing platform. Sequences were initially verified through one of two criteria; either a match between a de novo assembly and a reference mapping, or a match between all of five different reference mappings performed against a fixed set of starting reference genomes with significant genetic distances within the same species of viruses. All reference mappings used an iterative method to avoid bias. Further verification was achieved through phylogenetic analysis against published SVDV genomes and additional Enterovirus B sequences. This approach allows high confidence in the obtained consensus sequences, as well as provides sufficiently high and evenly dispersed sequence coverage to allow future studies of intra-host variation.
authors.affiliation: Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Sandra C Abel
authors.initials: SC
authors.lastname: Nielsen
authors.affiliation: Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Christian A W
authors.initials: CA
authors.lastname: Bruhn
authors.affiliation: Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Jose Alfredo
authors.initials: JA
authors.lastname: Samaniego
authors.affiliation: Vesicular Disease Reference Laboratory Group, The Pirbright Institute, Pirbright, Surrey, United Kingdom.
authors.forename: Jemma
authors.initials: J
authors.lastname: Wadsworth
authors.affiliation: Vesicular Disease Reference Laboratory Group, The Pirbright Institute, Pirbright, Surrey, United Kingdom.
authors.forename: Nick J
authors.initials: NJ
authors.lastname: Knowles
authors.affiliation: Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark.
authors.forename: M Thomas P
authors.initials: MT
authors.lastname: Gilbert
mesh_terms: D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D029822:Enterovirus B, Human / Q000235:genetics*; D016679:Genome, Viral / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D016013:Likelihood Functions; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary