Validation of CRC and NSCLC somatic mutations detected by NGS using ddPCR
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 diagno...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
| Published: |
University of Sarajevo, Institute for Genetic Engineering and Biotechnology
2019-06-01
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| Series: | Genetics & Applications |
| Subjects: | |
| Online Access: | https://genapp.ba/editions/index.php/journal/article/view/59 |
| Summary: | 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.
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| ISSN: | 2566-2937 2566-431X |