Figure 1.
Coverage profiles of one Norovirus sample from amplicon and direct RNA sequencing.
A – Coverage across the genome for one Norovirus sample sequenced from PCR amplicons (others similar). Green and orange dotted lined mark the locations of the PCR primers used to generate the amplicons. B – coverage across the genome for the same Norovirus sample sequenced directly from RNA.
Figure 2.
Coverage across the genome for two Hepatitis C samples sequenced directly from RNA.
Figure 3.
Evolutionary tree created by BEAST (Bayesian evolutionary analysis sampling trees) depicting all the full genomic sequences with relatedness (61 sequences, excluding repeated pairs).
Clusters of genomes are visible among viruses sampled at similar points in time. Whole genome sequencing gives adequate resolution to distinguish potential divergent viral strains within the same time, as illustrated in clusters from January 2010, February 2011 and March 2011. WO = ward outbreak. Each node and branch has been coloured depicting the posterior probability supporting that clade calculated by Bayesian analysis (Dark Blue = 1 (high); Light Red = 0 (low)). Analysis was performed using BEAST v.1.7.5 combining two random number seed chains (10 million iterations each, saving 1 in 1000 iterations, with a 1 million iteration burn-in) using: HKY substitution; estimated frequency; strict clock; and constant population size coalescent tree prior. This maximum clade credibility tree was computed using TreeAnnotator v.1.7.5 and plotted with Figtree v.1.4.0.
Figure 4.
Comparison of the different fragmentation methods.
A) fragment size distribution of a library prepared using the standard fragmentation method (red) and a library prepared using the new fragmentation method (blue). B) the coverage across the genome for the standard fragmentation sample (red) and the new fragmentation sample (blue). Data has been scaled as the difference from the median coverage for both samples.
Table 1.
A comparison of workflows and consumable costs for various viral sequencing approaches.
Figure 5.
Schematic representation of different strategies for viral genome resequencing.
A) Total RNA library: all the RNA species present in the sample are sequenced, no assumption on which genome is present, B) Hybridisation capture of a mRNA library: a good reference genome is needed to design the probes for capture, C) PCR enrichment: the desired genome is amplified from cDNA, a reference genome is needed to design specific oligos. Red lines, genomes of interest; Blue segments, Illumina adapters; Black lines, other RNA species.