Oncomine Colon cfDNA Sequencing Protocol

An Oncomine Colon cfDNA Assay (Thermo Fisher Scientific) was used to generate libraries from the cfDNA, following the manufacturer’s instructions. The purity and concentration of the library were assessed using the Qubit dsDNA HS Assay kit and the Agilent 2100 Bioanalyzer, respectively. Unique index tags were added to each DNA fragment during library preparation. The Ion Chef System and the Ion 530 Kit-Chef were used for template preparation, followed by sequencing on the Ion S5 system using Ion 530 chips. A six-plex library pool was applied to the Ion 530 chip. We used a cfDNA panel covering 14 genes and 48 amplicons, including 240 hot spots (SNVs and short indels). The genes included in the panel were KRAS, TP53, APC, FBXW7, GNAS, MAP2K1, CTNNB1, ERBB2, PIK3CA, BRAF, EGFR, SMAD4, NRAS, and AKT1. Clean reads were mapped to the human reference genome (hg19). A variant caller was used to filter and call mutations in targeted regions of each gene [20 (link), 21 (link)]. The limit of detection for each variant (mutant allele frequency [MAF]) was 0.15%. The average coverage ranged from 20,000x to 50,000x.