Comparative Evaluation of Comprehensive DNA and RNA Sequencing Platforms with Subsequent Clinical Validation for...

Background/Objectives: Genomic alterations play a central role in diagnosis, prognostication, and therapeutic planning for hematolymphoid malignancies. At our tertiary care center, frontline genomic testing relies on optical genome mapping, karyotyping, and fluorescence in situ hybridization, with targeted next-generation sequencing (NGS) performed externally. To provide more comprehensive genomic profiling, we evaluated two large-panel NGS platforms and subsequently performed a clinical validation of the selected assay. Methods: The Illumina PanHeme DNA panel and the SOPHiA Genetics Community Myeloid Solution were compared using 24 bone marrow aspirate specimens with previously characterized alterations, including single nucleotide variants (SNVs), insertions/deletions (indels), and copy number variants (CNVs). The selected panel underwent full analytical validation using 60 specimens. Results: Both panels demonstrated excellent concordance for SNVs and indels, with comparable analytical performance and workflow. CNV calling with SOPHiA was notably strong. Platform selection was influenced by practical considerations, including panel content and cost, leading to a preference for further evaluation of the Illumina assay. Clinical validation of the Illumina PanHeme DNA panel, along with a complementary RNA Exome panel, was subsequently performed. Sequence variant detection showed 100% concordance with orthogonal testing, while CNV detection was variable, reflecting known limitations of targeted NGS. The RNA panel detected all expected fusion transcripts. Conclusions: These findings demonstrate robust analytical performance of both evaluated DNA panels. Clinical validation of the Illumina PanHeme DNA and RNA Exome assays supports their use for comprehensive molecular profiling of hematologic malignancies.