E220 focused ultrasonicator

The tumor samples used for WES analysis were formalin-fixed paraffin-embedded (FFPE). Patient-matched peripheral blood samples were stored in Streck blood collection tubes and used as a normal control for germline filtering. DNA was extracted from the FFPE samples using the MagMAX FFPE DNA/RNA Ultra kit (cat# A31881, ThermoFisher, Waltham, MA, USA) and from the peripheral whole blood samples using the Maxwell RSC blood DNA kit (cat# AS1400, Promega, Madison, WI, USA). The recovered DNA was sheared to approximately ~200 bp using a Covaris E220 Focused-ultrasonicator and then hybridized to whole exome probes from the Agilent SureSelect XT Human All Exon V7 kit (cat# 5991-9039, Agilent, Santa Clara, CA, USA). After enrichment, the captured DNA was amplified, end-repaired, and attached to sequencing adapters and barcodes using the SureSelect XT HS and Low Input Library Preparation Kit for ILM (Pre-PCR) (cat# G9704, Agilent, Santa Clara, CA, USA). The libraries were normalized, pooled, and loaded on the Illumina NovaSeq-6000 Sequencing System to generate 150×150-bp paired-end reads. Raw image analysis and base-calling were carried out using Illumina onboard RTA3 program with default parameters.