Sample processing and DNA extraction
All fresh samples were stored in -80℃ refrigerators immediately till all
sample collection was finished. Peripheral blood was collected in EDTA
Vacutainer tubes (BD Diagnostics, Franklin Lakes, NJ, USA) and processed
within 4 h. Peripheral blood
lymphocytes (PBLs) from the first centrifugation were used for the
extraction of germline genomic DNA. The genomic DNA of frozen tumor
tissue and matched peripheral blood lymphocytes samples were isolated
using a commercially available DNeasy Blood & Tissue Kit (Qiagen,
Hilden, Germany). The genomic DNA of FFPE samples were isolated using
Maxwell® RSC DNA FFPE Kit (Promega, Madison, WI, USA). The DNA
concentration was measured using a Qubit fluorometer and the Qubit dsDNA
HS (High Sensitivity) Assay Kit (Invitrogen, Carlsbad, CA, USA). The
total DNA yield must be greater than or equal to 1 µg,while 260/280 and
260/230 is greater than or equal to 1.8 and 2, respectively.