Nanopore Sequencing Accurate in IGH Monitoring for CLL

An alternative approach to clonotyping immunoglobulin heavy chain (IGH) gene arrangements in patients with chronic lymphocytic leukemia (CLL) can produce highly accurate results, according to a new report.

The authors of the new report said nanopore sequencing produced results comparable to short-read next-generation sequencing (NGS) platforms, though they acknowledged that the newer technology still has limitations. The study was published in the European Journal of Haematology.¹

Corresponding author Marcus Høy Hansen, MSc, PhD, of Denmark’s Odense University Hospital, and colleagues said clonal rearrangements of IGH or T-cell receptor loci are important markers for disease monitoring in CLL, lending insights for disease classification and measurable residual disease assessment, among other uses.

Over time, they explained, the technology used in clonotyping has evolved from Southern blots, fragment analysis, and Sanger sequencing. However, the current state-of-the-art technology is NGS. Yet, Hansen and colleagues said the current short-read NGS platforms have significant limitations.

“Although short-read NGS platforms have provided unprecedented resolution, their limitations include challenges in spanning full variable-diversity-joining (VDJ) gene rearrangements and reduced base quality at the ends,” they wrote.

Strategies such as paired-end sequencing with overlapping reads can improve accuracy and help circumvent the problem, they said.

However, an alternate approach is Oxford Nanopore Technologies (ONT) sequencing, which the authors noted can produce long reads with real-time sequencing features. Previous research has suggested that nanopore sequencing is a feasible alternative to Sanger sequencing in clonotyping CLL.²

In the new report, the investigators sought to compare the new ONT long-read sequencing platform with short-read sequencing. The investigators took IGH clonotyping samples from 13 patients with CLL and analyzed them using the ONT MinION Mk1c nanopore sequencing device along with ONT’s single-use Flongle flow cells and super-accuracy graphics processing unit (GPU)-based Dorado base calling. For comparison, they also analyzed samples using the Illumina MiSeq-based LymphoTrack NGS assays.

Hansen and colleagues said all major and minor clonotypes identified using MiSeq were matched with nanotype sequencing. In terms of clonal burden estimates, there was a strong correlation but also considerable variability, they said (Pearson P = 0.87; P < 10⁻⁴).

“These results confirm that high-accuracy nanopore base calling can support clonotyping applications comparable to those performed on Illumina’s MiSeq platform,” they wrote.

Hansen and colleagues added that somatic hypermutation status was "reliably assessed” using super-accuracy base calling (Q30: 76%).

“This level of accuracy is sufficient to determine the mutational status of IGHV genes, a crucial prognostic marker in CLL,” they said.

The investigators said the ONT system has a smaller laboratory footprint and lower acquisition cost, making it an attractive choice for laboratories with limited resources. However, they also noted that the sequencing depth per Flongle flow cell is less than that of larger devices using standard flow cells, and they cited high variability in sequencing yield as another potential limitation.

Hansen and colleagues said more work should be done to optimize sequencing protocols and base-calling algorithms in order to refine the nanopore approach. They added that their study size was small, as their study was intended to serve as a proof of concept. They said larger cohorts will be needed for validation. In the meantime, though, they said their initial findings are promising.

“In summary, nanopore sequencing, combined with GPU-based high-accuracy base calling, provides reliable IGH clonotyping with performance comparable to MiSeq sequencing while maintaining a simple and flexible workflow,” they concluded.

References:

1. Hansen MH, Gulmir R, Cédile O, et al. Performance of rapid clonotyping of chronic lymphocytic leukemia using Flongle flow-cell nanopore sequencing of IGH rearrangements. Eur J Haematol. Published online September 8, 2025. doi:10.1111/ejh.70030

2. Minervini CF, Cumbo C, Redavid I, et al. Nanopore sequencing approach for immunoglobulin gene analysis in chronic lymphocytic leukemia. Sci Rep. 2021;11(1):17668. doi:10.1038/s41598-021-97198-3