Whole genome, deep sequencing of Hepacivirus C: UK retreatment patient
Libraries were prepared for Illumina sequencing using the NEBNext Ultra Directional RNA Library Prep Kit (New England Biolabs) with 8 microlitres of RNA extracted from plasma using NUCLISENS easyMAG (bioMrieux) and previously published modifications of the manufacturers guidelines (v2.0), briefly: omission of heat fragmentation, omission of Actinomycin D at first-strand reverse transcription, library amplification for 15 PCR cycles using custom indexed primers , and post-PCR clean-up with 0.85x volume Ampure XP (Beckman Coulter). Libraries were quantified using Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen) and size distribution analysed using Agilent TapeStation D1K High Sensitivity kit. A 500ng aliquot of the pooled libraries (96 plex) was enriched using the xGen Lockdown protocol (Rapid Protocol for DNA Probe Hybridization and Target Capture Using an Illumina TruSeq or Ion Torrent Library v1.0, Integrated DNA Technologies) with equimolar-pooled 120nt DNA oligonucleotide probes (Integrated DNA Technologies) followed by a 12-cycle on-bead post-enrichment PCR. The cleaned post-enrichment ve-Seq library was quantified by qPCR with the KAPA SYBR FAST qPCR Kit (Kapa Biosystems) and sequenced with 150b paired-end reads on a single run of the Illumina MiSeq.