Long-read sequencing and T2T-CHM13 enable accurate detection of complex pathogenic inversions in Duchenne muscular dystrophy

Conventional genetic testing fails to diagnose a subset of Duchenne muscular dystrophy (DMD) cases due to complex structural variants (SVs). We applied long-read sequencing (LRS) and RNA-seq to four DMD probands who tested negative on routine testing. LRS identified previously unreported long-range inversions (4–80 Mbp) disrupting the DMD locus in all probands. In one proband, alignment to the T2T-CHM13 reference remove the false positive calls when aligning to GRCh38, and RNA-seq revealed a pathogenic DMD - PRRG1 fusion transcript. All breakpoints were Sanger validated, with three inversions maternally inherited. Together, these results demonstrate that LRS with T2T-CHM13 was a powerful approach for resolving complex pathogenic SVs in DMD .