Targeted long-read sequencing for high-resolution repeat profiling in myotonic dystrophy type 1

Abstract Tandem repeat expansion disorders can be difficult to diagnose when expansions exceed 200 repeats, as standard methods (for example, Southern blot and modified PCR) often fail. We present a Cas9-targeted nanopore sequencing workflow and an automated analysis pipeline, RepeatLab, for accurate repeat-length estimation, structure assessment, and high-resolution methylation profiling. Validated on 13 myotonic dystrophy type 1 samples, 4 healthy controls, and 4 cell lines, this approach demonstrates improved sensitivity and accuracy for large expansions. Key refinements include an alternative basecalling strategy for extended repeats and a repeat-length calling algorithm that remains robust at lower sequencing throughput. The platform also automatically reports methylation near the DMPK repeat region, including five CpG site groups that could inform more nuanced clinical evaluations. This integrated workflow offers a rapid, cost-effective diagnostic solution with a turnaround time under 24 h and costs comparable to standard assays. Its compatibility with readily available computational resources enhances accessibility and scalability.