4.9. Analysis of Circulating Tumor DNA (ctDNA)
NRAS
COLON, APC
DNA was extracted from 3 mL of plasma using a QIAmp Circulating Nucleic Acid kit (Qiagen) according to the manufacturer’s instructions. Identification of mutations in 14 genes (AKT1, APC, BRAF, CTNNB1, EGFR, ERBB2, FBXW7, GNAS, KRAS, MAP2K1, NRAS, PIK3CA, SMAD4, TP53) was performed in parallel using the Oncomine Colon cfDNA assay on the Ion S5 sequencing platform. Briefly, starting with 10 ng of cfDNA, manual libraries were prepared following manufacturer instructions. Libraries were quantified using qPCR, and 80 pM of each library (24 per chip) was loaded onto a 540 chip and sequenced. Sequencing data were analyzed using the Oncomine Colon Liquid Biopsy—w1.4—DNA—Single Sample workflow. Samples with at least an average coverage depth of 10,000 passed sequencing QC. For mutations, the following filters were used: variant coverage ≥ 10, variant frequency ≥ 0.1%, and 