Quality control material for the detection of somatic mutations in fixed clinical specimens by next-generation sequencin
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RESEARCH
Open Access
Quality control material for the detection of somatic mutations in fixed clinical specimens by next-generation sequencing Catherine I. Dumur*, Jorge A. Almenara, Celeste N. Powers and Andrea Ferreira-Gonzalez
Abstract Background: Targeted next generation sequencing (NGS) technology to assess the mutational status of multiple genes on formalin-fixed, paraffin embedded (FFPE) tumors is rapidly being adopted in clinical settings, where quality control (QC) practices are required. Establishing reliable FFPE QC materials for NGS can be challenging and/or expensive. Here, we established a reliable and cost-effective FFPE QC material for routine utilization in the Ion AmpliSeq™ Cancer Hotspot Panel v2 (CHP2) assay. Methods: The performance characteristics of the CHP2 assay were determined by sequencing various cell line mixtures and 55 different FFPE tumors on the Ion Torrent PGM platform. A FFPE QC material was prepared from a mixture of cell lines derived from different cancers, comprising single nucleotide variants and small deletions on actionable genes at different allelic frequencies. Results: The CHP2 assay performed with high precision and sensitivity when custom variant calling pipeline parameters where established. In addition, all expected somatic variants in the QC material were consistently called at variant frequencies ranging from 9.1 % (CV = 11.1 %) to 37.9 % (CV = 2.8 %). Conclusions: The availability of a reliable and cost-effective QC material is instrumental in assessing the performance of this or any targeted NGS assay that detects somatic variants in fixed solid tumor specimens.
Background The numerous cancer genome characterization efforts that have emerged in the past years [1–3] have promoted the development of targeted cancer therapeutics [4], including single or combined inhibitory agents [5], which reportedly are beneficial to individuals who have tumors harboring specific somatic mutations in genes encoding for proteins involved in cell growth, proliferation, and survival signaling pathways [6–9]. Thus, molecular testing to identify such mutations in clinical specimens to assess patient eligibility for targeted therapies has become standard practice in the management of oncology patients. The recent technological advances in next-generation sequencing (NGS) and the applications in the field of oncology are revolutionizing clinical testing for personalized treatment decisions for oncology patients [10]; as well as the translational research field, where somatic * Correspondence: [email protected] Department of Pathology, Virginia Commonwealth University, Clinical Support Center, Room 247, 403 North 13th Street, Richmond, VA 23298, USA
variant findings may enhance the development of novel targeted cancer therapeutics, which could benefit individuals with tumors harboring such mutations. A variety of different NGS-based assays have been developed for mutation identification that have the ability to detect all mutation types, including single nucleotide varian
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