Qubit 3.0 with dsdna hs assay kit

Genomic DNAs from FFPE samples and the whole blood control samples were extracted using Qiagen QIAamp DNA FFPE Tissue Kit and DNeasy Blood and tissue kits (Qiagen, USA)), respectively, and quantified using Qubit 3.0 with dsDNA HS Assay Kit (ThermoFisher Scientific, USA). Sequencing library preparation was performed with KAPA Hyper Prep Kit (KAPA Biosystems, USA). DNA libraries were pooled and captured with a custom 425 cancer-gene panel. The capture reaction was performed with Dynabeads M- 270 (Life Technologies, CA, USA) and xGen Lockdown hybridization and wash kit (Integrated DNA Technologies) according to manufacturers’ protocols. Captured libraries were PCR amplified with KAPA HiFi HotStartReadyMix (KAPA Biosystems), followed by purification using AgencourtAMPure XP beads. Libraries were quantified by qPCR using KAPA Library Quantification kit (KAPA Biosystems). Library fragment size was determined by Bioanalyzer 2100 (Agilent Technologies). The target-enriched library was then sequenced on HiSeq4000 NGS platforms (Illumina) to a minimum coverage depth of 100X and 600X for blood and FFPE, respectively. Exome capture was performed using the IDT xGen Exome Research Panel V1.0 (Integrated DNA Technologies) and sequenced using HiSeq4000 to a mean coverage depth of ~60X for the normal control (white blood cells samples) and ~150X for the tumor FFPE samples.