Abstract
BackgroundThe detection of somatic mutations in primary tumors is critical for the understanding of cancer evolution and targeting therapy. Multiple technologies have been developed to enable the detection of such mutations. Next generation sequencing (NGS) is a new platform that is gradually becoming the technology of choice for genotyping cancer samples, owing to its ability to simultaneously interrogate many genomic loci at massively high efficiency and increasingly lower cost. However, multiple barriers still exist for its broader adoption in clinical research practice, such as fragmented workflow and complex bioinformatics analysis and interpretation.MethodsWe performed validation of the QIAGEN GeneReader NGS System using the QIAact Actionable Insights Tumor Panel, focusing on clinically meaningful mutations by using DNA extracted from formalin-fixed paraffin-embedded (FFPE) colorectal tissue with known KRAS mutations. The performance of the GeneReader was evaluated and compared to data generated from alternative technologies (PCR and pyrosequencing) as well as an alternative NGS platform. The results were further confirmed with Sanger sequencing.ResultsThe data generated from the GeneReader achieved 100% concordance with reference technologies. Furthermore, the GeneReader workflow provides a truly integrated workflow, eliminating artifacts resulting from routine sample preparation; and providing up-to-date interpretation of test results.ConclusionThe GeneReader NGS system offers an effective and efficient method to identify somatic (KRAS) cancer mutations.
Highlights
The detection of somatic mutations in primary tumors is critical for the understanding of cancer evolution and targeting therapy
We recently evaluated the QIAGEN GeneReader System workflow from Deoxyribonucleic acid (DNA) extraction and purification from formalin-fixed paraffin-embedded (FFPE) tissue samples, to library preparation, sequencing and data analysis and interpretation
Evaluation of DNA quality by QuantiMIZE FFPE samples with ages ranging from 3 to 20 years were used for this study
Summary
The detection of somatic mutations in primary tumors is critical for the understanding of cancer evolution and targeting therapy. Multiple technologies have been developed to enable the detection of such mutations. Somatic mutations in the KRAS oncogene are common in human cancers. Sanger sequencing has been the ‘gold standard’ for mutation analysis in cancer detection since the 1970s [11]. The therascreen KRAS RGQ PCR kit is a real-time qPCR-based assay used to detect the most common KRAS mutations including those in codons 12 and 13. It has greatly improved sensitivity over Sanger sequencing, and has been approved by the Food and Drug Administration (FDA) [9] for colorectal cancer patient stratification
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