2.2. Blood samples, ctDNA isolation and sequencing

Blood samples were collected into EDTA tubes following the manufacturer’s instructions. Plasma from blood sample was obtained by centrifugation at 1600 g for 10 minutes at 4°C, followed by another spin at 16 000 g for 10 minutes at 4°C to remove cellular debris. cfDNA was extracted from 2 mL plasma using a MagMAX cfDNA Isolation Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. Oncomine Colon cfDNA Assay (Thermo Fisher Scientific) was used to generate libraries from cfDNA following the manufacturer’s instructions. Quality control of the libraries was performed using the Qubit^®2.0 and 2100 Bioanalyzer (Agilent Technologies). Ion Chef™ System and Ion 530™ Kit‐Chef were used for template preparation, followed by sequencing on Ion S5 system using Ion 530 chips. Six‐plex library pool was applied on an Ion 530 chip. The cfDNA panel, which covers 14 genes with >240 hot spots (SNVs and short Indels), such as AKT1, BRAF, CTNNB1, EGFR, ERBB2, FBXW7, GNAS, KRAS, MAP2K1, NRAS, PIK3CA, SMAD4, TP53, and APC, was used in this study. The clean reads were mapped to the human reference genome (hg19). Variant caller was used to filter and call the mutations in targeted regions in each gene.²⁴, ²⁵ Cutoff level for each mutant allele frequency was defined by “variant caller” for each sample (patient). The range of the limit of detection for the variants in KRAS and NRAS was from 0.05 to 0.20 in this study.