title: Comparison of Next-Generation Sequencing Panels and Platforms for Detection and Verification of Somatic Tumor Variants for Clinical Diagnostics.
abstract: Use of next-generation sequencing to detect somatic variants in DNA extracted from formalin-fixed, paraffin-embedded tumor tissues poses a challenge for clinical molecular diagnostic laboratories because of variable DNA quality and quantity, and the potential to detect low allele frequency somatic variants difficult to verify by non-next-generation sequencing methods. We evaluated somatic variant detection performance of the MiSeq and Ion Proton benchtop sequencers using two commercially available panels, the TruSeq Amplicon Cancer Panel and the AmpliSeq Cancer Hotspot Panel Version 2. Both the MiSeq-TruSeq Amplicon Cancer Panel and Ion Proton-AmpliSeq Cancer Hotspot Panel Version 2 were comparable in terms of detection of somatic variants and allele frequency determination using DNA extracted from tumor tissue. Concordance was 100% between the panels for detection of somatic variants in genomic regions tested by both panels, including 27 variants present at low somatic allele frequency (<15%). Use of both the MiSeq and Ion Proton platforms in a combined workflow enabled detection of potentially actionable variants with importance for patient diagnosis, prognosis, or treatment in 49% (305/621) of cases. Overall, a combined workflow using both platforms enabled successful molecular profiling of 96% (621/644) of tumor samples, and provided an approach for verification of somatic variants not amenable to verification by Sanger sequencing (<15% variant allele frequency).
authors: Advanced Molecular Diagnostics Laboratory, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.
authors: Maksym
authors: M
authors: Misyura
authors: Advanced Molecular Diagnostics Laboratory, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.
authors: Tong
authors: T
authors: Zhang
authors: Advanced Molecular Diagnostics Laboratory, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.
authors: Mahadeo A
authors: MA
authors: Sukhai
authors: Advanced Molecular Diagnostics Laboratory, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.
authors: Mariam
authors: M
authors: Thomas
authors: Advanced Molecular Diagnostics Laboratory, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.
authors: Swati
authors: S
authors: Garg
authors: Advanced Molecular Diagnostics Laboratory, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada; Department of Laboratory Genetics, Laboratory Medicine Program, University Health Network, Toronto, Ontario, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada; Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
authors: Suzanne
authors: S
authors: Kamel-Reid
authors: Advanced Molecular Diagnostics Laboratory, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada; Department of Laboratory Genetics, Laboratory Medicine Program, University Health Network, Toronto, Ontario, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada. Electronic address: tracy.stockley@uhn.ca.
authors: Tracy L
authors: TL
authors: Stockley
mesh_terms: D000483:Alleles; D014408:Biomarkers, Tumor; D005787:Gene Frequency; D005820:Genetic Testing / Q000379:methods / Q000592:standards; D014644:Genetic Variation; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods / Q000592:standards; D006801:Humans; D009369:Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D057188:Workflow
chemical_list: D014408:Biomarkers, Tumor