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Recurrent gene fusions are common drivers of disease pathophysiology in leukemias. Identifying these structural variants helps stratify disease by risk and assists with therapy choice. Precise molecular diagnosis in low-and-middle-income countries (LMIC) is challenging given the complexity of assays, trained technical support, and the availability of reliable electricity. Current fusion detection methods require a long turnaround time (7-10 days) or advance knowledge of the genes involved in the fusions. Recent technology developments have made sequencing possible without a sophisticated molecular laboratory, potentially making molecular diagnosis accessible to remote areas and low-income settings. We describe a long-read sequencing DNA assay designed with CRISPR guides to select and enrich for recurrent leukemia fusion genes, that does not need a priori knowledge of the abnormality present. 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::AFF1) 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. The platform's portability, potential for adaptation to lower-cost devices, and integrated cloud analysis make this assay a candidate to be placed in settings like LMIC to bridge the need of bedside rapid molecular diagnostics., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: LHH and KYY have equity interest in Biodepot LLC, which receives compensation from NCI SBIR contract numbers 75N91020C00009 and 75N91021C00022. The terms of this arrangement have been reviewed and approved by the University of Washington in accordance with its policies governing outside work and financial conflicts of interest in research. This work is related to the following two provisional patent applications: “Optimized assay and efficient computational workflow to deliver same-day leukemia diagnostics” (patent application 63/416,888) “Detecting Leukemia Specific Gene Fusions” (patent application 63/309261), (Copyright: © 2023 Sala-Torra et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Chronic myelogenous leukemia (CML) is a hematopoietic stem cell malignancy that accounts for 15-20% of all cases of leukemia. CML is caused by a translocation between chromosomes 9 and 22 which creates an abnormal fusion gene, BCR::ABL1. The amount of BCR::ABL1 transcript RNA is a marker of disease progression and the effectiveness of tyrosine kinase inhibitor (TKI) treatment. This study determined the analytical and clinical performance of a droplet digital PCR based assay (QXDx BCR-ABL %IS Kit; Bio-Rad) for BCR::ABL1 quantification. The test has a limit of detection of MR4.7 (0.002%) and a linear range of MR0.3-4.7 (50-0.002%IS). Reproducibility of results across multiple sites, days, instruments, and users was evaluated using panels made from BCR::ABL1 positive patient samples. Clinical performance of the assay was evaluated on patient samples and compared to an existing FDA-cleared test. The reproducibility study noted negligible contributions to variance from site, instrument, day, and user for samples spanning from MR 0.7-4.2. The assay demonstrated excellent clinical correlation with the comparator test using a Deming regression with a Pearson R of 0.99, slope of 1.037 and intercept of 0.1084. This data establishes that the QXDx™ BCR-ABL %IS Kit is an accurate, precise, and sensitive system for the diagnosis and monitoring of CML., Competing Interests: Bio-Rad provided reagents to each site to support this study. D.N.S, P.B., N.S., and S.H. are employees of Bio-Rad. Bio-Rad employees have or may have stock in Bio-Rad. In addition to providing employment, the stated Bio-Rad employees have or may have patents, products in development or marketed products in Bio-Rad. This does not alter our adherence to PLOS ONE policies on sharing data and materials.