Development and Validation of StrataNGS, a Multiplex PCR, Semiconductor Sequencing-Based Comprehensive Genomic Profiling Test

Despite widespread use in targeted tumor testing, multiplex PCR/semiconductor (Ion Torrent) sequencing-based assessment of all comprehensive genomic profiling (CGP) variant classes has been limited. Herein, we describe the development and validation of StrataNGS, a 429-gene, multiplex PCR/semiconductor sequencing-based CGP laboratory-developed test performed on co-isolated DNA and RNA from formalin-fixed, paraffin-embedded tumor specimens with ≥2 mm2 tumor surface area. Validation was performed in accordance with MolDX CGP validation guidelines using 1986 clinical formalin-fixed, paraffin-embedded samples and an in-house developed optimized bioinformatics pipeline. Across CGP variant classes, accuracy ranged from 0.945 for tumor mutational burden (TMB) status to >0.999 for mutations and gene fusions, positive predictive value ranged from 0.915 for TMB status to 1.00 for gene fusions, and reproducibility ranged from 0.998 for copy number alterations to 1.00 for splice variants and insertions/deletions. StrataNGS TMB estimates were highly correlated to those from whole exome– or FoundationOne CDx–determined TMB (Pearson r = 0.998 and 0.960, respectively); TMB reproducibility was 0.996 (concordance correlation coefficient). Limit of detection for all variant classes was