RNA was extracted using Roche High Pure viral RNA kits. Water only controls were extracted, amplified and sequenced in parallel with each set of samples. Capsid coding regions were amplified in duplicate by one-step RT-PCR using a SuperScript III HiFi kit and primers P1F (5’-GCGAGTTGGATTGGCCATCCAGTG -3’) and P1R (5’-TGGAAGGTGGGTCCCACAAACGAC-3’). Products were purified using AMPure XP magnetic beads (Beckman Coulter), quantified using Qubit High Sensitivity dsDNA assay (Life Technologies), analysed on an Agilent High Sensitivity DNA chip (Agilent) and diluted to 0.2 ng/μl in molecular grade 10mM Tris–EDTA, pH8.0.
Sequencing libraries were prepared using Nextera XT reagents (Illumina) and the manufacturer's protocol, and sequenced on a MiSeq using a 2 × 251 paired-end v2 Flow Cell (Illumina). Quality trimming and assembly were carried out as in Mee at al. [26 (link)]. Reads were then mapped to parental reference sequences using Geneious R7 (Biomatters) software and SNPs present at ≥ 0.5% identified. Only those SNPs present in both replica amplicons were retained.
Sequencing libraries were prepared using Nextera XT reagents (Illumina) and the manufacturer's protocol, and sequenced on a MiSeq using a 2 × 251 paired-end v2 Flow Cell (Illumina). Quality trimming and assembly were carried out as in Mee at al. [26 (link)]. Reads were then mapped to parental reference sequences using Geneious R7 (Biomatters) software and SNPs present at ≥ 0.5% identified. Only those SNPs present in both replica amplicons were retained.
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