Assessment of long-read strategies for the enrichment of clinically relevant breakpoints in lymphomas: towards a diagnostic implementation

TL;DR

The study compares different long-read sequencing methods to detect genetic breakpoints in lymphomas, aiming to improve diagnostic accuracy and treatment.

Contribution

A Cas9-based enrichment panel and decision algorithm are proposed for optimal breakpoint detection in clinical lymphoma diagnostics.

Findings

Cas9-mediated enrichment provides superior on-target coverage for densely targeted regions like the IGH locus.

Adaptive sampling offers higher throughput and flexibility but has limited breakpoint detection capabilities.

Cas9 excision is a fast and reliable method for detecting canonical translocation partners in clinical samples.

Abstract

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.

The online version contains supplementary material available at 10.1007/s00277-026-06754-2.

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