title: Whole genome, deep sequencing of Hepacivirus C: UK retreatment patient
design: RNA was extracted from 350 microlitres of plasma using the NUCLISENS easyMAG system (bioMrieux). Total eluates were subjected to Turbo DNAse treatment (Thermo Fisher) followed by library preparation using KAPA RNA HyperPrep kit (Roche). Libraries were pooled based upon DNA concentration and HCV quantity, assessed using the Quant-iT kit on the Glomax platform (Promega) and the Qiagen QuantiTect kit with primers and probes from Davalieva et al. 2014. Pools were enriched by hybridisation to a biotinylated probe set (Integrated DNA Technologies, described in Bonsall et al. 2015) followed by further PCR cycles depending upon HCV quantity.  The two pools were pooled, again by concentration and HCV quantity. The final pool was quantified using the KAPA SYBR FASTA qPCR kit (Roche) on a PRISM 7500 (Applied BioSystems) before being sequenced on a MiSeq using Reagent kit v2 (both Illumina).