Recurrent chromosomal translocations are hallmarks of many hematological malignancies, including lymphomas and leukemias. Accurate breakpoint detection is essential for diagnostics, treatment optimization, and disease monitoring. Long-read sequencing (Oxford Nanopore Technologies) enables unambiguous mapping and translocation identification. We designed a Cas9-based enrichment panel targeting common translocations in lymphoid malignancies. To accommodate both well-defined and promiscuous translocation partners, we employed single-side and dual-side sequencing strategies. Using well-established lymphoid cell lines, we benchmarked three enrichment approaches: (i) Cas9 read-out, (ii) Cas9 excision with multiplexing, and (iii) adaptive sampling. Cas9-mediated enrichment achieved superior on-target coverage, particularly in densely targeted regions (such as the IGH locus), while single-probe targets showed lower coverage depth. Adaptive sampling offered higher throughput, flexibility, and better pore occupancy, however with limited breakpoint detection. Cas9 excision has been demonstrated as a fast and reliable method to detect canonical translocation partners in clinical lymphoma samples. Our findings indicate that long-read enrichment strategies are suitable for targeting breakpoint hotspots, although the choice of approach depends heavily on the laboratory's specific goal. We propose a decision algorithm for selecting the optimal method based on experimental and clinical needs: Cas9-mediated enrichment suits focused diagnostic intent, while adaptive sampling is preferable for broader research use.

Center of Molecular Medicine CEITEC Central European Institute of Technology Masaryk University Brno Czech Republic

Centre of Molecular Biology and Genetics Department of Internal Medicine Hematology and Oncology University Hospital Brno and Faculty of Medicine Masaryk University Brno Czech Republic

Institute of Medical Genetics and Genomics Faculty of Medicine Masaryk University and University Hospital Brno Brno Czech Republic

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