A pilot study on the application of Oxford Nanopore PromethION sequencing with adaptive sampling for DMPK repeat expansion sizing in myotonic dystrophy type 1

Abstract

Myotonic dystrophy type 1 (DM1) is caused by a CTG repeat expansion in the DMPK gene, but conventional polymerase chain reaction and Southern blot-based workflows provide limited resolution for large expansions and somatic mosaicism. This pilot study evaluated Oxford Nanopore PromethION sequencing with adaptive sampling for DMPK repeat expansion sizing. Fifteen samples, including Coriell reference materials, European Molecular Genetics Quality Network external quality assessment materials, and clinical samples, were sequenced across seven adaptive-sampling runs using native barcoding. DMPK repeat-spanning reads were aligned to the reference genome and summarized at read level. Sequencing runs generated a median of 38.9 gigabases of data and 56.2 million reads, with median DMPK repeat-locus coverage of 49×. Reducing multiplexing to two samples per flow cell improved median expanded-allele coverage. Normal allele sizing was concordant across reference, external quality assessment, and clinical samples, and expanded-allele estimates in reference materials and selected clinical samples were consistent with prior orthogonal results. Single-molecule reads also provided repeat-size distributions consistent with intra-sample variability. However, expanded-allele coverage remained variable, and low-read observations limited confidence for some samples. These findings support adaptive-sampling PromethION sequencing as a feasible pilot approach for DMPK repeat expansion sizing, while indicating that improved coverage and larger clinical validation are needed before routine diagnostic implementation.