Recurrent gene fusions are common drivers of disease pathophysiology in leukemias. Identification of these structural variants helps stratify disease by risk and assists with therapy choice. Current fusion detection methods require long turnaround time (7-10 days) or advance knowledge of the genes involved in the fusions. To address the need for rapid identification of clinically actionable fusion genes in heme malignancies without a-priori knowledge of the genes, we describe a long-read sequencing DNA assay designed with CRISPR guides to select and enrich for recurrent leukemia fusion genes. By applying rapid sequencing technology based on nanopores, we sequenced long pieces of genomic DNA and successfully detected fusion genes in cell lines and primary specimens (e.g., BCR-ABL1, PML-RARA, CBFB-MYH11, KMT2A-AF4) using cloud-based bioinformatics workflows with novel custom fusion finder software. We detected fusion genes in 100% of cell lines with the expected breakpoints and confirmed the presence or absence of a recurrent fusion gene in 12 of 14 patient cases. With our optimized assay and cloud-based bioinformatics workflow, these assays and analyses could be performed in under 8 hours.

LHH and KYY also have equity interest in Biodepot LLC, which receives compensation from NCI SBIR contract numbers 75N91020C00009 and 75N91021C00022.

This study was supported in part by NCCN young investigator award and the Hyundai Hope on Wheels Scholars Award, R01 CA175008-06, UG1 CA233338-02, and Adult Leukemia Research Center Grant # P01 CA018029 as funded by the National Cancer Institute, National Institutes of Health, Bethesda, MD. LHH, SR and KYY are supported by NIH grant R01GM126019. SR is also supported by the Vicky L. Carwein and William B. Andrews Endowments for Graduate Programs.

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All samples used in this study were deidentified discarded samples stored in a biorepository with Fred Hutchison Cancer Center IRB approval. The Fred Hutchinson Cancer Center IRB gave ethical approval for this work.

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Data from this study are available upon request to the authors.