Validation of CRC and NSCLC somatic mutations detected with NGS using ddPCR
Journal Title: Genetics & Applications - Year 2019, Vol 3, Issue 1
Abstract
Next Generation Sequencing (NGS) has become powerful tool in molecular oncology. It allows multiparallel targeted sequencing that enables comprehensive assessment of tumor heterogeneity. Detection of mutations in colorectal cancer (CRC) and non-small cell lung cancer (NSCLC) defines patients diagnosis, therapy and prognosis. Multiple genes, their somatic mutations to be precise, carry different degrees of importance for each of these aspects. Ion AmpliSeq™ Colon and Lung Cancer Research Panel v2, which was used in this study, allows detection of hotspot mutations in 22 genes in a single reaction. Droplet digital PCR (ddPCR) has a unique advantage in low frequency mutation detection and it has been used as a validation tool for mutations that were detected with NGS. It has high sensitivity and enables accurate detection of a mutant allele against a background of abundant wild type alleles. For this study 35 samples of CRC and NSCLC were sequenced and selected samples were analysed with ddPCR for KRAS, NRAS, EGFR and BRAF genes. All the processed samples were successfully sequenced and had average base coverage >500X. NGS sequencing proved itself to be cost effective, has shorter turnaround time and is highly sensitive. Out of 35 samples, 25 had genetic alterations, while 10 samples were reported as wild type but were still tested with ddPCR as controls. In three samples low frequency somatic mutations were detected with NGS and mutation frequencies were verified using ddPCR.
Authors and Affiliations
Lana Salihefendić, Dino Pećar, Rijad Konjhodžić
Keywords
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- EP ID EP592367
- DOI 10.31383/ga.vol3iss1pp71-76
- Views 113
- Downloads 0
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