title: Quality control material for the detection of somatic mutations in fixed clinical specimens by next-generation sequencing.
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.
authors.affiliation: Department of Pathology, Virginia Commonwealth University, Clinical Support Center, Room 247, 403 North 13th Street, Richmond, VA, 23298, USA. catherine.dumur@vcuhealth.org.
authors.forename: Catherine I
authors.initials: CI
authors.lastname: Dumur
authors.affiliation: Department of Pathology, Virginia Commonwealth University, Clinical Support Center, Room 247, 403 North 13th Street, Richmond, VA, 23298, USA. jorge.almenara@vcuhealth.org.
authors.forename: Jorge A
authors.initials: JA
authors.lastname: Almenara
authors.affiliation: Department of Pathology, Virginia Commonwealth University, Clinical Support Center, Room 247, 403 North 13th Street, Richmond, VA, 23298, USA. celeste.powers@vcuhealth.org.
authors.forename: Celeste N
authors.initials: CN
authors.lastname: Powers
authors.affiliation: Department of Pathology, Virginia Commonwealth University, Clinical Support Center, Room 247, 403 North 13th Street, Richmond, VA, 23298, USA. andrea.ferreira-gonzalez@vcuhealth.org.
authors.forename: Andrea
authors.initials: A
authors.lastname: Ferreira-Gonzalez
mesh_terms: D016477:Artifacts; D014408:Biomarkers, Tumor / Q000235:genetics*; D045744:Cell Line, Tumor; D003362:Cost-Benefit Analysis; D004252:DNA Mutational Analysis / Q000191:economics / Q000592:standards*; D005404:Fixatives; D005557:Formaldehyde; D005787:Gene Frequency; D020022:Genetic Predisposition to Disease; D017048:Health Care Costs; D059014:High-Throughput Nucleotide Sequencing / Q000191:economics / Q000592:standards*; D006801:Humans; D057230:Limit of Detection; D009154:Mutation; D009369:Neoplasms / Q000235:genetics*; D016612:Paraffin Embedding; D020641:Polymorphism, Single Nucleotide; D011237:Predictive Value of Tests; D011786:Quality Control; D015203:Reproducibility of Results; D016707:Tissue Fixation
chemical_list: D014408:Biomarkers, Tumor; D005404:Fixatives; D005557:Formaldehyde