Library preparation and sequencing
Genomic DNA from a xenograft derived pancreatic ductal adenocarcinoma cell-line and matching normal sample was combined in eight different proportions (100, 60, 40, 20, 15, 10, 5 and 0 % tumour), each totalling 3 ?g. Qubit (Life Technologies, Carlsbad, CA, USA Cat #Q32854) was used to quantify the cell-line and normal gDNA. The mixed gDNA was sheared to 200 bp fragments using the Covaris S2 Ultra-sonicator (Covaris Inc, Woburn, MA, USA) and a 1× volume AMPure XP SPRI bead clean-up (Beckman Coulter Genomics, Danvers, MA, USA Cat #A63881) was applied. Illumina paired-end libraries were prepared on the Sciclone NGS workstation (Perkin Elmer, Waltham, MA, USA) using the NEBNext DNA Sample Prep Master Mix Set for Illumina (New England Biolabs, Ipswich, MA, USA Cat #E6000) and IDT oligos (Integrated DNA Technologies, San Jose, CA, USA). From the purified library, 500 ng was used as input for a 72 h hybridization at 65 °C to Agilent Human SureSelect All Exon (v4) baits (Agilent Technologies, Santa Clara, CA, USA Cat #5190-4632). Targeted DNA was recovered using Dynabeads MyOne Streptavidin T1 (Life Technologies, Carlsbad, CA, USA Cat #65601).
Libraries were validated using the Agilent Bioanalyzer High Sensitivity DNA Kit (Agilent Technologies, Santa Clara, CA, USA Cat #5067-4626) and quantified on the Illumina Eco Real-Time PCR Instrument (Illumina Inc., San Diego, CA, USA) using KAPA Illumina Library Quantification Kits (KAPA Biosciences, Woburn, MA, USA Cat# KK4835) according to the standard manufacturer’s protocols. Paired-end cluster generation (Illumina Inc., San Diego, CA, USA Cat #PE-401-3001) and sequencing of 2 × 101 cycles (Illumina Inc., San Diego, CA, USA Cat #FC-401-3001) was carried out for all eight libraries on the Illumina Hi-Seq 2000 platform (Illumina Inc., San Diego, CA, USA), one library per lane.