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http://hdl.handle.net/1942/26514
Title: | Validation and Application of a Custom-Designed Targeted Next-Generation Sequencing Panel for the Diagnostic Mutational Profiling of Solid Tumors | Authors: | FROYEN, Guy Broekmans, An Hillen, Femke Pat, Karin ACHTEN, Ruth MEBIS, Jeroen RUMMENS, Jean-Luc Willemse, Johan MAES, Brigitte |
Issue Date: | 2016 | Source: | PLOS ONE, 11(4) | Abstract: | The inevitable switch from standard molecular methods to next-generation sequencing for the molecular profiling of tumors is challenging for most diagnostic laboratories. However, fixed validation criteria for diagnostic accreditation are not in place because of the great variability in methods and aims. Here, we describe the validation of a custom panel of hotspots in 24 genes for the detection of somatic mutations in non-small cell lung carcinoma, colorectal carcinoma and malignant melanoma starting from FFPE sections, using 14, 36 and 5 cases, respectively. The targeted hotspots were selected for their present or future clinical relevance in solid tumor types. The target regions were enriched with the TruSeq approach starting from limited amounts of DNA. Cost effective sequencing of 12 pooled libraries was done using a micro flow cell on the MiSeq and subsequent data analysis with MiSeqReporter and VariantStudio. The entire workflow was diagnostically validated showing a robust performance with maximal sensitivity and specificity using as thresholds a variant allele frequency >5% and a minimal amplicon coverage of 300. We implemented this method through the analysis of 150 routine diagnostic samples and identified clinically relevant mutations in 16 genes including KRAS (32%), TP53 (32%), BRAF (12%), APC (11%), EGFR (8%) and NRAS (5%). Importantly, the highest success rate was obtained when using also the low quality DNA samples. In conclusion, we provide a workflow for the validation of targeted NGS by a custom-designed pan-solid tumor panel in a molecular diagnostic lab and demonstrate its robustness in a clinical setting. | Document URI: | http://hdl.handle.net/1942/26514 | ISSN: | 1932-6203 | e-ISSN: | 1932-6203 | DOI: | 10.1371/journal.pone.0154038 | ISI #: | 000374898500129 | Rights: | : © 2016 Froyen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. | Category: | A1 | Type: | Journal Contribution |
Appears in Collections: | Research publications |
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