DUCKS4: a comprehensive workflow for Nanopore sequencing analysis of facioscapulohumeral muscular dystrophy (FSHD).

MOTIVATION: Facioscapulohumeral Muscular Dystrophy (FSHD) is an autosomal dominant form of muscular dystrophy caused by genetic or epigenetic changes within the D4Z4-repeat at the DUX4-gene, on chromosome 4q. Genetic analysis is challenging due to a nearly identical region on chromosome 10, multiple haplotypes, long and short repeat subtypes, and complex rearrangements such as translocations and duplications. So far, no single method detects all known causes of FSHD.

RESULTS

We have developed an integrated approach combining an optimized wet-lab protocol with an automated bioinformatics workflow, called DUCKS4. It enables read-level resolution of the D4Z4 array for FSHD1 repeat sizing, variant detection for FSHD2, and detection of methylation patterns. Using NCBI BLAST, it assigns reads to chromosomes and haplotypes, supporting robust filtering and analysis. Our approach also includes a novel adaptive sampling workflow for human high molecular-weight DNA. With long-read Nanopore sequencing technology, our tool enables precise determination of D4Z4 array size, individual haplotype assignment, methylation profiling, and complex allele analysis. It also allows for the detection of mosaicism and structural variation like interchromosomal translocations, providing a comprehensive, single-method solution for FSHD analysis. AVAILABILITY AND IMPLEMENTATION: DUCKS4 is implemented within a Docker environment. Source code and supplementary materials are accessible at https://github.com/tamara-nano/ducks4 , https://github.com/tamara-nano/ducks4_wovar (light version without variant calling) and archived at https://figshare.com/projects/DUCKS4-FSHD/260306