# 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.

## Key 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.