Translating Sanger-Based Routine DNA Diagnostics into Generic Massive Parallel Ion Semiconductor Sequencing

摘要: BACKGROUND: Dideoxy-based chain termination sequencing developed by Sanger is the gold standard approach and allows clinical diagnostics of disorders with relatively low genetic heterogeneity. Recently, new next generation (NGS) technologies have found their way into diagnostic laboratories, enabling large targeted gene panels or exomes. The development benchtop NGS instruments now analysis single genes small panels, making these platforms increasingly competitive sequencing. METHODS: We a generic automated ion semiconductor work flow that can be used in setting serve as substitute for sequencing. Standard amplicon-based enrichment remained identical to PCR A novel postenrichment pooling strategy was developed, limiting number library preparations reducing costs up 70% compared sequencing. RESULTS: total 1224 known pathogenic variants were analyzed, yielding an analytical sensitivity 99.92% specificity 99.99%. In second experiment, 100 patient-derived DNA samples analyzed using blind analysis. results showed 99.60% 99.98%, comparable sequencing. CONCLUSIONS: Ion first choice mutation scanning technique, independent analyzed.