Validation of CRC and NSCLC somatic mutations detected by NGS using ddPCR

Abstract

Next Generation Sequencing (NGS) has become powerful tool in molecularoncology. It allows multiparallel targeted sequencing that enablescomprehensive assessment of tumor heterogeneity. Detection of mutations incolorectal cancer (CRC) and non-small cell lung cancer (NSCLC) definespatients diagnosis, therapy and prognosis. Multiple genes, their somaticmutations to be precise, carry different degrees of importance for each of theseaspects. Ion AmpliSeq™ Colon and Lung Cancer Research Panel v2, which wasused in this study, allows detection of hotspot mutations in 22 genes in a singlereaction. Droplet digital PCR (ddPCR) has a unique advantage in low frequencymutation detection and it has been used as a validation tool for mutations thatwere detected with NGS. It has high sensitivity and enables accurate detection ofa mutant allele against a background of abundant wild type alleles. For this study35 samples of CRC and NSCLC were sequenced and selected samples wereanalysed with ddPCR for KRAS, NRAS, EGFR and BRAF genes. All theprocessed samples were successfully sequenced and had average base coverage>500X. NGS sequencing proved itself to be cost effective, has shorterturnaround time and is highly sensitive. Out of 35 samples, 25 had geneticalterations, while 10 samples were reported as wild type but were still tested withddPCR as controls. In three samples low frequency somatic mutations weredetected with NGS and mutation frequencies were verified using ddPCR.