Supporting Data for: Performance of rapid clonotyping of chronic lymphocytic leukemia using Flongle flow-cell Nanopore sequencing of IGH rearrangements

Background: Clonotyping of immunoglobulin heavy chain (IGH) gene rearrangements is critical for diagnosis, prognostication, and measurable residual disease (MRD) monitoring in chronic lymphocytic leukemia (CLL). While short-read next-generation sequencing (NGS) platforms like Illumina MiSeq are widely used, they face challenges in spanning full VDJ rearrangements. Flexible sequencing via Oxford Nanopore Technologies (ONT) offers a potential alternative, especially using the compact and cost-effective Flongle flow cell. Methods: We evaluated IGH clonotyping in 13 CLL patients using ONT MinION with Flongle flow cells and super-accuracy GPU-based base calling (Dorado), comparing results to MiSeq-based LymphoTrack assays. Amplicons were generated using the IGH FR1 assay and analyzed using Smith-Waterman alignment and IgBlast tools. Results: All clonotypes identified by MiSeq were matched with 100% sequence identity using Flongle. Clonal burden estimates were strongly correlated (Pearson ρ = 0.86, p < 10⁻⁴), and somatic hypermutation status was reliably assessed with super-accuracy base calling. Fast or CPU-only base calling was insufficient for accurate mutation analysis. Conclusion: Nanopore Flongle sequencing enables accurate and rapid IGH clonotyping in CLL, offering comparable performance to MiSeq with low cost and laboratory footprint. This supports its potential utility in routine and decentralized hematopathology workflows.