Qubit fluorometer with the dsdna high sensitivity assay kit

After collection, peripheral blood samples were processed within 72 h to separate the plasma from the peripheral blood cells, and transferred to fresh tubes for storage at −80°C until DNA isolation. DNA isolation and subsequent sequencing procedures were performed in the laboratory of Burning Rock Biotech (Guangzhou, China) accredited and certified by the CAP and Clinical Laboratory Improvement Amendments (CLIA). Circulating cell-free DNA (cfDNA) were extracted using QIAamp Circulating Nucleic Acid Kits (Qiagen, Hilden, Germany) from 0.5–2.0 mL of the plasma samples. Genomic DNA (gDNA) used as normal control were extracted from white blood cells (WBCs) by QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). Qubit fluorometer with the dsDNA high-sensitivity assay kit (Life Technologies, Carlsbad, CA, USA) was used to measure DNA quality following the manufacturer's instructions.

DNA isolation and subsequent sequencing procedures were performed in the laboratory of Burning Rock Biotech (Guangzhou, China) accredited and certified by the College of American Pathologists and Clinical Laboratory Improvement Amendments. Genomic DNA (gDNA) was extracted from tumor tissues and white blood cells by using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). Qubit fluorometer with the dsDNA high-sensitivity assay kit (Life Technologies, Carlsbad, CA, United States) was used to measure DNA quality following the manufacturer’s instructions. DNA fragmentation was performed using a Covaris M220 Focused-ultrasonicator (Woburn, MA, United States), followed by end repair, phosphorylation, and adapter ligation. Fragments between 200 and 400 bp were selected using AMPure beads (Agencourt AMPure XP Kit, Beckman Coulter, Brea, CA, United States). Subsequently, the hybridization with capture probe baits, hybrid selection with magnetic beads, and PCR amplification were performed. A high-sensitivity DNA assay was then performed to assess the quality and size of the DNA fragments (Agilent 2,100 bioanalyzer instrument, Agilent, Santa Clara, CA, United States).