European journal of haematology · 2025
Performance of Rapid Clonotyping of Chronic Lymphocytic Leukemia Using Flongle Flow-Cell Nanopore Sequencing of IGH Rearrangements.
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Hansen Marcus Høy, Gulmir Rona, Cédile Oriane, Dahlmann Sara Kamuk, Veyhe Sólja Remisdóttir, Stougaard Magnus, Juul-Jensen Karen, Nyvold Charlotte Guldborg
Аннотация
BACKGROUND: Clonotyping of immunoglobulin heavy chain (IGH) gene rearrangements is critical for diagnosis, prognostication, and measurable residual disease monitoring in chronic lymphocytic leukemia (CLL). Although short-read next-generation sequencing (NGS) platforms, such as Illumina MiSeq, are widely used, they face challenges in spanning full VDJ rearrangements. Long-read sequencing via Oxford Nanopore Technologies (ONT) offers a potential alternative using the compact and cost-effective flow cells.
METHODS: We evaluated IGH clonotyping in samples from 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 major and minor clonotypes identified by MiSeq were matched with 100% sequence identity using Nanopore sequencing. Clonal burden estimates were strongly correlated (Pearson ρ = 0.87, p < 10-4), although with considerable variability. Somatic hypermutation status was reliably assessed with super-accuracy base calling (Q30: 76%). Fast or CPU-only base calling was insufficient for accurate mutation analysis.
CONCLUSION: Nanopore sequencing enables accurate and rapid IGH clonotyping in CLL, offering comparable performance to MiSeq with lower cost and laboratory footprint. This supports its potential utility in routine and decentralized hematopathology workflows.
METHODS: We evaluated IGH clonotyping in samples from 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 major and minor clonotypes identified by MiSeq were matched with 100% sequence identity using Nanopore sequencing. Clonal burden estimates were strongly correlated (Pearson ρ = 0.87, p < 10-4), although with considerable variability. Somatic hypermutation status was reliably assessed with super-accuracy base calling (Q30: 76%). Fast or CPU-only base calling was insufficient for accurate mutation analysis.
CONCLUSION: Nanopore sequencing enables accurate and rapid IGH clonotyping in CLL, offering comparable performance to MiSeq with lower cost and laboratory footprint. This supports its potential utility in routine and decentralized hematopathology workflows.