title: Target-Capture sequencing of felis catus: adult male liver tumour infected with DCHBV
design: FFPE liver-derived DNA was available from 16 HCC biopsies that tested DCHBV-positive by PCR and 4 DCHBV-negative HCCs. A total of 5 ng of total nucleic acid extract was used to prepare libraries using the EF Library preparation kit (version 1, Twist Biosciences, CA, USA) and dual indexes (TruSeq compatible Twist UMI Adapter System, Twist Biosciences). Minor modifications were made to the manufactures library preparation protocol. Briefly, fragmentation time was reduced to 2 minutes and 15 seconds, and a total of 16 amplification cycles was used during library amplification. DNA library concentration was assessed using the Qubit dsDNA High Sensitivity Quantitation Assay (Thermo Fisher Scientific) on the Qubit 4 Fluorometer (Thermo Fisher Scientific) and up to 96 libraries were pooled in a total of 2 ml for multiplex hybridisation. Library pools were dried down for 17 hours prior to standard hybridisation with the Twist Comprehensive Viral Research Panel (Twist Biosciences). This panel contains probes that capture the complete genome of 3,153 viruses, including the DCHBV genome. Following post-capture amplification and purification, captured libraries were quantified using the Qubit dsDNA High Sensitivity Quantitation Assay (Thermo Fisher Scientific) on Qubit 4 Fluorometer (Thermo Fisher Scientific) and library fragment size was assessed using the Bioanalyzer High Sensitivity D5000 ScreenTapes (Agilent). The molarity of each capture pool was determined and, if required, additional post capture pools were combined in equimolar concentrations prior to sequencing using 300 cycle Illumina sequencing chemistry. Sequencing was performed with an expected 1.5 million reads per library.