<div><p>Somatic mutations in <i>KRAS</i>, <i>NRAS</i>, and <i>BRAF</i> genes are related to resistance to anti-EGFR antibodies in colorectal cancer. We have established an extended <i>RAS</i> and <i>BRAF</i> mutation assay using a next-generation sequencer to analyze these mutations. Multiplexed deep sequencing was performed to detect somatic mutations within <i>KRAS</i>, <i>NRAS</i>, and <i>BRAF</i>, including minor mutated components. We first validated the technical performance of the multiplexed deep sequencing using 10 normal DNA and 20 formalin-fixed, paraffin-embedded (FFPE) tumor samples. To demonstrate the potential clinical utility of our assay, we profiled 100 FFPE tumor samples and 15 plasma samples obtained from colorectal cancer patients. We used a variant calling approach based on a Poisson distribution. The distribution of the mutation-positive population was hypothesized to follow a Poisson distribution, and a mutation-positive status was defined as a value greater than the significance level of the error rate (α = 2 x 10<sup>-5</sup>). The cut-off value was determined to be the average error rate plus 7 standard deviations. Mutation analysis of 100 clinical FFPE tumor specimens was performed without any invalid cases. Mutations were detected at a frequency of 59% (59/100). <i>KRAS</i> mutation concordance between this assay and Scorpion-ARMS was 92% (92/100). DNA obtained from 15 plasma samples was also analyzed. <i>KRAS</i> and <i>BRAF</i> mutations were identified in both the plasma and tissue samples of 6 patients. The genetic screening assay using next-generation sequencer was validated for the detection of clinically relevant <i>RAS</i> and <i>BRAF</i> mutations using FFPE and liquid samples.</p></div
