Use of the QIAGEN GeneReader NGS system for detection of KRAS mutations, validated by the QIAGEN Therascreen PCR kit and alternative NGS platform

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-017-3328-z) contains supplementary material, which is available to authorized users.

Why Formalin-fixed, Paraffin-embedded Biospecimens Must Be Used in Genomic Medicine: An Evidence-based Review and Conclusion

“…In another study in which the UDG-containing GeneRead DNA extraction method was compared with a non-UDG-containing extraction method, C:G > T:A substitution differences only became apparent when the allele frequency cutoff was reduced from 5% to 2.5%, at which point the call was correct when extractions had been performed using the GeneRead kit. 18 A further study found that up to 94% of the observed C:G > T:A substitutions present in FFPE samples were artifactual and could be reversed using UDG the treatment, but the frequency was so low that there was “little impact on sensitivity.” 19 …”

A Next-Generation Sequencing Primer—How Does It Work and What Can It Do?

“…Test results can be helpful for the initial diagnosis, tumor classification, determining the origin of the cancer, and prognosis. 76 Table 2 77 – 130 provides a partial list of cancers where NGS information has provided value for managing patients. Thyroid nodules are a specific example where fine needle aspiration and cytologic examination may not yield a definitive diagnosis, while NGS has been shown to have high specificity and sensitivity for cancer detection.…”

Use of the Ion PGM and the GeneReader NGS Systems in Daily Routine Practice for Advanced Lung Adenocarcinoma Patients: A Practical Point of View Reporting a Comparative Study and Assessment of 90 Patients

“…The introduction of targeted therapies into the clinical treatment of patients with advanced lung adenocarcinoma has made routine testing of tumor mutations a daily task in molecular pathology laboratories. The Qiagen GeneReader system is the first sequencing system commercially available that spans the whole workflow from nucleic acid isolation to data analysis, with the potential to replace currently used “homebrew” workflows for the genetic analysis, as previously demonstrated in comparison to Illumina MiSeq sequencing, Sanger sequencing and pyrosequencing; systems which are commonly used for genetic assessment in routine molecular pathology [ 15 , 16 ].…”