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Near-complete genome sequencing of swine vesicular disease virus using the Roche GS FLX sequencing platform.

Nielsen SC, Bruhn CA, Samaniego JA, Wadsworth J, Knowles NJ, Gilbert MT - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark.

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.

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Phylogenetic validation of samples.Using a maximum likelihood approach as described in Methods on the 1B-1C-1D genome region, corresponding to the outer capsid proteins VP2, VP3 and VP1, final validation was obtained for the samples. The five Hong Kong SVDVs isolated in the 1970s and which were sequenced in this study, are shown in red and all other SVD virus isolates are shown in pink. Coxsackievirus B5 isolates are shown in blue, and other Enterovirus B serotypes are shown in black. As expected from the previous literature, all CV-B5 together with all SVDV form a monophyletic cluster. This is supported with a bootstrap proportion of 100/100. Within this cluster all the SVD virus isolates, including those sequenced in this study, form a monophyletic cluster with bootstrap support of 96/100. Additionally, none of the five presently sequenced Hong Kong isolates show any obvious aberrations with regards to their position in the topology concerning either geographical information or branch lengths (vs. age). The tree is rooted on the branch leading from CV-B5 to all other CV-B sequences, and all branch labels show support in the form of bootstrap proportions out of one hundred. Support values for nodes with minor importance regarding the verification are not shown.
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pone-0097180-g002: Phylogenetic validation of samples.Using a maximum likelihood approach as described in Methods on the 1B-1C-1D genome region, corresponding to the outer capsid proteins VP2, VP3 and VP1, final validation was obtained for the samples. The five Hong Kong SVDVs isolated in the 1970s and which were sequenced in this study, are shown in red and all other SVD virus isolates are shown in pink. Coxsackievirus B5 isolates are shown in blue, and other Enterovirus B serotypes are shown in black. As expected from the previous literature, all CV-B5 together with all SVDV form a monophyletic cluster. This is supported with a bootstrap proportion of 100/100. Within this cluster all the SVD virus isolates, including those sequenced in this study, form a monophyletic cluster with bootstrap support of 96/100. Additionally, none of the five presently sequenced Hong Kong isolates show any obvious aberrations with regards to their position in the topology concerning either geographical information or branch lengths (vs. age). The tree is rooted on the branch leading from CV-B5 to all other CV-B sequences, and all branch labels show support in the form of bootstrap proportions out of one hundred. Support values for nodes with minor importance regarding the verification are not shown.

Mentions: At nearly three times the length of alignments typically employed for VP1 based phylogenies, our alignment can be considered highly informative. The phylogenetic tree (Figure 2), rooted on the branch between the CV-B5's and all other CV-B's, shows all CV-B5 sequences and all SVDV sequences forming a highly supported (bootstrap proportion 100/100) monophyletic cluster, with the SVDV sequences themselves forming a highly supported (bootstrap proportion 96/100) monophyletic cluster within this. This conforms to the currently accepted evolutionary relationship of the strains for this genomic region. Furthermore, the topology shows no apparent aberrations with regards to the geographical location or age of isolates vs. branch lengths with regards to the five SVDV samples.


Near-complete genome sequencing of swine vesicular disease virus using the Roche GS FLX sequencing platform.

Nielsen SC, Bruhn CA, Samaniego JA, Wadsworth J, Knowles NJ, Gilbert MT - PLoS ONE (2014)

Phylogenetic validation of samples.Using a maximum likelihood approach as described in Methods on the 1B-1C-1D genome region, corresponding to the outer capsid proteins VP2, VP3 and VP1, final validation was obtained for the samples. The five Hong Kong SVDVs isolated in the 1970s and which were sequenced in this study, are shown in red and all other SVD virus isolates are shown in pink. Coxsackievirus B5 isolates are shown in blue, and other Enterovirus B serotypes are shown in black. As expected from the previous literature, all CV-B5 together with all SVDV form a monophyletic cluster. This is supported with a bootstrap proportion of 100/100. Within this cluster all the SVD virus isolates, including those sequenced in this study, form a monophyletic cluster with bootstrap support of 96/100. Additionally, none of the five presently sequenced Hong Kong isolates show any obvious aberrations with regards to their position in the topology concerning either geographical information or branch lengths (vs. age). The tree is rooted on the branch leading from CV-B5 to all other CV-B sequences, and all branch labels show support in the form of bootstrap proportions out of one hundred. Support values for nodes with minor importance regarding the verification are not shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016283&req=5

pone-0097180-g002: Phylogenetic validation of samples.Using a maximum likelihood approach as described in Methods on the 1B-1C-1D genome region, corresponding to the outer capsid proteins VP2, VP3 and VP1, final validation was obtained for the samples. The five Hong Kong SVDVs isolated in the 1970s and which were sequenced in this study, are shown in red and all other SVD virus isolates are shown in pink. Coxsackievirus B5 isolates are shown in blue, and other Enterovirus B serotypes are shown in black. As expected from the previous literature, all CV-B5 together with all SVDV form a monophyletic cluster. This is supported with a bootstrap proportion of 100/100. Within this cluster all the SVD virus isolates, including those sequenced in this study, form a monophyletic cluster with bootstrap support of 96/100. Additionally, none of the five presently sequenced Hong Kong isolates show any obvious aberrations with regards to their position in the topology concerning either geographical information or branch lengths (vs. age). The tree is rooted on the branch leading from CV-B5 to all other CV-B sequences, and all branch labels show support in the form of bootstrap proportions out of one hundred. Support values for nodes with minor importance regarding the verification are not shown.
Mentions: At nearly three times the length of alignments typically employed for VP1 based phylogenies, our alignment can be considered highly informative. The phylogenetic tree (Figure 2), rooted on the branch between the CV-B5's and all other CV-B's, shows all CV-B5 sequences and all SVDV sequences forming a highly supported (bootstrap proportion 100/100) monophyletic cluster, with the SVDV sequences themselves forming a highly supported (bootstrap proportion 96/100) monophyletic cluster within this. This conforms to the currently accepted evolutionary relationship of the strains for this genomic region. Furthermore, the topology shows no apparent aberrations with regards to the geographical location or age of isolates vs. branch lengths with regards to the five SVDV samples.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark.

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.

Show MeSH
Related in: MedlinePlus