Results of assemblies and mappings of five Hong Kong swine vesicular disease virus (SVDV) isolates.

<p>First row shows the names of the five isolates, passage history, and harvest date. Second row shows the number of reads assigned to each isolate after they were separated using MID barcodes, and the number of these that had unique sequences (i.e. with duplicates removed), which were then kept for assembly/mapping. A number of reads is also lost during de-multiplexing, however this is even smaller than the total number of duplicates (not shown). Row three shows the mean, the standard deviation and the maximum of the read lengths of the unique reads. The fourth row shows results from <i>de novo</i> assembly, including number of reads mapped and minimum, maximum, mean, and standard deviation figures for the depth of coverage of the contig produced. The following five rows show results from iterative mapping against the five selected starting reference sequences (two SVDV sequences, a coxsackievirus B5, a coxsackievirus B3, and a coxsackievirus B6 sequence). The three isolates with successful <i>de novo</i> assemblies were iteratively mapped against one or two of these sequences for validation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097180#pone-0097180-g001" target="_blank">Figure 1</a>), whereas the remaining two isolates were iteratively mapped against all five starting references. For each of these two isolates the final mapping statistics can be seen to be virtually identical across all five starting references, clearly indicating that convergence of the iterative mapping process has been obtained. The final row shows the total number of ‘polymorphic’ sites obtained when the consensus sequence from assembly (if successful) and all of the mappings are multiple-aligned for each isolate (in parenthesis is shown whether any of the ‘polymorphisms’ are due to base calls where a non-redundant call in one sequence matches the category of a redundant call in the other).</p

Decision diagram for validation of consensus sequences.

<p>The diagram illustrates the logic of the applied methodology for obtaining consensus sequences validated for further analysis. The process begins with <i>de novo</i> assembly as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097180#s2" target="_blank">methods</a> section. The starting point from there is the blue-outline box in the top left hand corner. Positive answers follow the green ‘YES’ arrows, negative ones follow the red ‘NO’ arrows, and grey arrows are followed in all cases. Termination in a red box should lead to thorough analysis of upstream sources of error, including everything from contaminated samples to late stage <i>in silico</i> problems. Arriving at the first green box means that the consensus sequence of assembly/mapping has been verified. Arriving at the final green box means that the sample sequence is fully validated, now also with regard to sample provenance. The sample sequence is now ready to be used for further scientific analysis.</p

Phylogenetic validation of samples.

<p>Using a maximum likelihood approach as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097180#s2" target="_blank">Methods</a> 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 <i>Enterovirus B</i> 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.</p

SAT 2 Phylogenetic Tree.

<p>Markov Chain Monte Carlo phylogenetic tree generated using the combined SAT 2 data set including both chronologically sampled (CHR) data sets and temporal sample clusters (CLU). Temporal sample clusters are collapsed and coloured. Posterior probabilities are given for each node and the scale bar indicates a branch length corresponding to 25 years.</p

SAT 1 Phylogenetic Tree.

<p>Markov Chain Monte Carlo phylogenetic tree generated using the combined SAT 1 data set including both chronologically sampled (CHR) data sets and temporal sample clusters (CLU). Temporal sample clusters are collapsed and coloured. Posterior probabilities are given for each node and the scale bar indicates a branch length corresponding to 25 years.</p

<i>dN/dS</i> ratios for all datasets.

<p>Single likelihood ancestor counting (SLAC) P < 0.1. Serotype sequences constituted 221 amino acids (SAT 1), 216 amino acids (SAT 2).</p><p><i>dN/dS</i> ratios for all datasets.</p

Visualization of the 24 overlapping PCR products representing the complete FMDV genome generated from the loading pad (LP) from LFD loaded with UKG 7B/2007.

<p>1.8% agarose gel stained with ethidium bromide (0.2 µg per ml). Lane 1, S-fragment of the 5′UTR, Lanes 2–22 L fragment, Lanes 23 and 24, poly A fragment.</p

Ct values generated from FMDV 3D rRT-PCR performed on sections of 5 separate LFDs and MagNA pure extracted RNA from the original epithelial suspensions and recorded by time of LFD testing.

<p>LP: Loading Pad; WS: Wicking strip; NB: Nitrocellulose below Ab Band (NB); Nitrocellulose Ab Band (AbB); Nitrocellulose above Ab band (NA). LFDs spanned two serotypes O (red dots) (LFDs HKN 10/2005, UKG 7B/2007) and A (blue dots) (LFDs TUR 20/2006, IRN53/2006, IRN 36/2007).</p

Ct values generated from FMDV 3D rRT-PCR performed on sections of 14 separate LFDs and corresponding values generated for MagNA pure extracted RNA from the equivalent original epithelial suspensions in parallel.

<p>LP: Loading Pad; WS: Wicking strip; NB: Nitrocellulose below Ab Band (NB); Nitrocellulose Ab Band (AbB); Nitrocellulose above Ab band (NA). LFDs spanned four serotypes O (red dots) (LFDs TUR 8/1969, BAR 2/2008, KUW 2/2008, SAU 3/2008, ZAM 5/2008, HKN 10/2005, IRN 53/2006, UKG 7B/2007, A (blue dots) (LFDs BAR 4/2009, IRN 1/2008, KEN 8/2008, TUR 20/2006, IRN 36/2007), Asia 1 (grey dots) (IRN 15/2001) and SAT 1 (yellow dots) (ZAM 5/2008).</p

Box plot of Ct values generated from FMDV 3D rRT-PCR performed on sections of LFDs from serotype SAT 1 isolate (ZAM 5/2008) eluted at one month after LFD testing.

<p>LFDs had either been stored at room temperature (RoomT) (green bar) or 37°C (red bar). LP: Loading Pad; WS: Wicking strip; Nitrocellulose Ab Band (AbB). Dotted blue line represents baseline data (Ct values from LFDs washed on day one).</p