Ion Torrent based multi-gene sequencing as a reliable diagnostic assay for the detection of somatic mutations in tumors using minimal DNA amounts from FFPE material

Targeted Next Generation Sequencing (NGS) represent a suitable way to implement parallel testing of multiple genetic aberrations, which is necessary for individualizing cancer treatment. Technically NGS can be implemented with increasing ease, however challenges with regard to input material and analysis of the data require extensive testing of the available technology platforms. This study aimed to determine whether the type of input material (formalin fixed paraffin embedded (FFPE) vs fresh frozen (FF) tumors) affected the outcome of NGS and which data analysis parameters should be used for reliable variant calling. The Ion Torrent PGM in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 was used to sequence frequently mutated regions of 50 genes. For 250 FFPE samples the detected mutations were confirmed using standard diagnostic assays. Moreover, we used Ion Torrent analysis software and constructed a standardized bio-informatics analysis pipeline supplementing the IT variant caller output with variant annotation and characterized 386 tumors using these settings. Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a concordance between NGS and conventional sequencing techniques of 98.5% where NGS provides the advantage of low input DNA concentration and detection of low-frequency variants. Concluding, targeted NGS can be reliably implemented in cancer diagnostics on both FFPE and FF tissue when using appropriate analysis settings.