Towards a Next-Generation Sequencing Diagnostic Service for Tumour Genotyping: A Comparison of Panels and Platforms.
Detection of clinically actionable mutations in diagnostic tumour specimens aids in the selection of targeted therapeutics. With an ever increasing number of clinically significant mutations identified, tumour genetic diagnostics is moving from single to multigene analysis. As it is still not feasible for routine diagnostic laboratories to perform sequencing of the entire cancer genome, our approach was to undertake targeted mutation detection. To optimise our diagnostic workflow, we evaluated three target enrichment strategies using two next-generation sequencing (NGS) platforms (Illumina MiSeq and Ion PGM). The target enrichment strategies were Fluidigm Access Array custom amplicon panel including 13 genes (MiSeq sequencing), the Oxford Gene Technologies (OGT) SureSeq Solid Tumour hybridisation panel including 60 genes (MiSeq sequencing), and an Ion AmpliSeq Cancer Hotspot Panel including 50 genes (Ion PGM sequencing). DNA extracted from formalin-fixed paraffin-embedded (FFPE) blocks of eight previously characterised cancer cell lines was tested using the three panels. Matching genomic DNA from fresh cultures of these cell lines was also tested using the custom Fluidigm panel and the OGT SureSeq Solid Tumour panel. Each panel allowed mutation detection of core cancer genes including KRAS, BRAF, and EGFR. Our results indicate that the panels enable accurate variant detection despite sequencing from FFPE DNA.
Yorkshire Regional Genetics Service, St. James's University Hospital, Leeds LS9 7TF, UK ; Leeds Institute of Cancer & Pathology, University of Leeds, St. James's University Hospital, Leeds LS9 7TF, UK.
George J
GJ
Burghel
Leeds Institute of Cancer & Pathology, University of Leeds, St. James's University Hospital, Leeds LS9 7TF, UK.
Carolyn D
CD
Hurst
Yorkshire Regional Genetics Service, St. James's University Hospital, Leeds LS9 7TF, UK ; Leeds Institute of Biomedical & Clinical Sciences, University of Leeds, St. James's University Hospital, Leeds LS9 7TF, UK.
Christopher M
CM
Watson
Leeds Institute of Cancer & Pathology, University of Leeds, St. James's University Hospital, Leeds LS9 7TF, UK.
Phillip A
PA
Chambers
Yorkshire Regional Genetics Service, St. James's University Hospital, Leeds LS9 7TF, UK.
Helen
H
Dickinson
Yorkshire Regional Genetics Service, St. James's University Hospital, Leeds LS9 7TF, UK.
Paul
P
Roberts
Leeds Institute of Cancer & Pathology, University of Leeds, St. James's University Hospital, Leeds LS9 7TF, UK.
Margaret A
MA
Knowles
D004273:DNA, Neoplasm / Q000235:genetics; D003954:Diagnostic Services; D005557:Formaldehyde / Q000737:chemistry; D052138:Genes, Neoplasm / Q000235:genetics; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D009154:Mutation / Q000235:genetics; D009369:Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D016612:Paraffin Embedding / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods
D004273:DNA, Neoplasm; D005557:Formaldehyde