Qiaamp circulating nuclear acid kit

cfDNA was extracted from 4–6 ml plasma using the QIAamp Circulating Nuclear Acid Kit (Qiagen) according to the manufacturer’s protocol. cfDNA concentration was measured using the Qubit 3.0 Fluorometer (ThermoFisher Scientific). Extracted DNA in the first several samples were run on the Bioanalyzer 2100 (Agilent Techonologies) to evaluate successful extraction of cfDNA. The DNA exhibited a peak at 168 bp which was consistent with that of cfDNA (Supplementary Fig. S7).
gDNA from WBC and tumor tissue was extracted using the DNeasy Blood and Tissue Kit (Qiagen) and the QIAamp DNA Mini Kit (Qiagen), respectively. gDNA concentration was measured using the Nanodrop 2000 spectrophotometer (ThermoFisher Scientific). cfDNA and gDNA were stored at −30 °C.

Cell‐free DNA (cfDNA) was isolated from plasma using the QIAamp Circulating Nuclear Acid Kit (QIAGEN, Venlo, Netherlands). Multiplex PCR was performed using a NEBNext Ultra II Q5 Master Mix (New England Biolabs, Inc., MA, USA). A library was constructed using the VAHTS® Universal DNA Library Prep Kit for Illumina V3 (Nanjing Vazyme Biotech Co. Ltd., Nanjing, China) and sequenced using a NovaSeq 6000 sequencer (Illumina Inc., CA, USA). For a SNP pool comprised of all mutations, two or more mutations were judged to be MRD positive, whereas one or no mutations were judged to be MRD negative.

EGFR mutations were initially detected via tissue biopsy using amplification refractory mutation system polymerase chain reaction (ARMS-PCR) analysis or next-generation sequencing.
Secondary T790M mutation analysis was performed using one of the following samples: biopsied tumor tissues, plasma, or CSF samples. Approximately 10 mL of whole blood and 10 mL of CSF (EDTA as the anticoagulant) were collected for the purpose of cfDNA extraction. The retrieved CSF was used for analysis within 4 h of the lumbar puncture procedure. Plasma and CSF were centrifuged at 1,600 × g and 10,000 × g, respectively, for 10 min at room temperature. CfDNA was extracted using the QiAamp circulating nuclear acid kit (Qiagen, Hilden, Germany). Droplet digital PCR (ddPCR) was used to detect cfDNA using the Sysmex OncoBeam EGFR kit (SYSMEX, Japan). All protocols of analysis were carried out in strict accordance with the manufacturer's instructions.