Analytical and Clinical Validation of Duoseq, a Novel Assay for Rapid, on-Site Clinical DNA and RNA Sequencing of Hematologic Malignancies

• Published in: Blood 2023-11, Vol.142 (Supplement 1), p.3670-3670
• Main Authors: Hsi, Eric D., Czader, Magdalena, Hill, Brian T., Thacker, Elizabeth, Wang, Lin, Giauque, Christopher, Vrydaghs, Andrea, Sperling, Matt, Bouldin, Jordan, Erno, Robert, Happ, Lanie, Parker, Clayton, Love, Cassandra L., Shingleton, Jennifer, Dave, Sandeep
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2023-189030

Introduction: Next generation sequencing (NGS) has become a critical component of the workup of malignancies. NGS can provide important diagnostic information including mutations, chromosomal copy number alterations and translocations (from DNAseq) as well as gene expression and fusions (from RNAseq). Incorporation of NGS in the clinic remains difficult. DNAseq and RNAseq remain distinct assays with challenging bioinformatics and turnaround times (TAT) that can last several weeks. Duoseq is a novel assay for combined DNA- and RNAseq in a single workflow that detects single nucleotide variants (SNVs), insertions/deletions (indels), splice site variants (SSVs), structural variants (SVs) and Epstein-Barr virus (EBV) status. Initially implemented for hematologic malignancies, Duoseq assesses nearly all known gene drivers (N=475) and is compatible with both fresh-frozen and formalin-fixed, paraffin-embedded (FFPE) tissues. It is available as a reagent kit and includes cloud-based bioinformatics, enabling any clinical lab with an Illumina sequencer to perform NGS in-house with a TAT of 2-3 days. Methods/Results: Duoseq was validated for concordance with commercial DNA- and RNAseq kits (New England BioLabs (NEB)). Sequencing libraries were prepared on DNA alone, RNA alone and total nucleic acid (TNA) from cell lines in accordance with kit protocols and captured using the Duoseq panel. Results were equivalent between Duoseq and individual (NEB) library preparations with nearly complete concordance in DNA variants (99.4%, P