Dneasy blood kit

Next-generation sequencing (NGS)-based somatic mutation were performed in a College of American Pathologists-accredited laboratory, Geneplus-Beijing (Beijing, China) from January 2017 to July 2020. All tissue samples included in this study underwent pathology review onsite to confirm histologic classification and the adequacy of the tumor tissues, which required a minimum of 20% of tumor cells. Genomic tumor DNA was extracted from the tumor tissues using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA). Genomic DNA was extracted from white blood cells as a germline control using the DNeasy Blood Kit (Qiagen, Valencia, CA, USA). DNA concentration was measured using a Qubit fluorometer and the Qubit dsDNA HS (High Sensitivity) Assay Kit (Invitrogen, Carlsbad, CA, USA). Sequencing libraries were prepared from Illumina TruSeq DNA Library Preparation Kits (Illumina, San Diego, CA, USA). Libraries were hybridized to custom-designed biotinylated oligonucleotide probes (NimbleGen SeqCap EZ Library, Roche NimbleGen, Madison, WI, USA), covering ~230 Kbp genomic regions of 59 genes or ~1.4 Mbp genomic regions of 1021 cancer-related genes (Supplemental Tables S1 and S2) using Gene + Seq 2000 instrument.^{37 (link),38 (link)}