Abstract
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.
Highlights
Genetic factors play a principal role in cancer predisposition, initiation, and development [1,2,3]
MiSeq cluster densities of 811 K/mm2 and 880 K/mm2 were achieved for the Fluidigm custom panel libraries prepared using fresh Genomic DNA (gDNA) and formalin-fixed paraffin-embedded (FFPE) gDNA, respectively
The per-sample read distribution is displayed in Supplementary Table 5 which indicates that the pooling was performed accurately
Summary
Genetic factors play a principal role in cancer predisposition, initiation, and development [1,2,3]. Throughout cancer progression, the genome acquires somatic genetic and epigenetic changes [4, 5]. Some mutations play a critical role in cancer development by affecting key cancer “driver” genes. Some changes do not provide any selective advantage and are termed “passenger” mutations [6]. A number of the “driver” gene mutations are considered “actionable” as they have diagnostic, prognostic, or predictive implications. Some of these mutations can be targeted by specific therapies and are commonly termed “druggable” variants [7]
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