Deciphering D4Z4 CpG methylation gradients in fascioscapulohumeral muscular dystrophy using nanopore sequencing

Cas9-targeted nanopore sequencing of D4Z4 repeats. (A) Schematic of 4q and 10q D4Z4 repeat arrays with a hypomethylated 5U array shown on the permissive 4qA haplotype. (B) Locations of Cas9-guide RNA cleavage sites at the centromeric (p13E-11) end, within the D4Z4 repeat, and at the telomeric (pLAM) end. Steps used in the sequencing pipeline for identifying targeted reads are shown. (C) Read depth Manhattan plots from two FSHD1 participants (P1, P5) mapped to the T2T CHM13 v2.0 reference genome (log₂ scale, minimum depth = 2 reads, summarized in 2 kb, nonoverlapping windows). Colors indicate reads mapped to the 4q and 10q D4Z4 arrays, as well as divergent DUX4 clusters. (D) Integrative Genomics Viewer (IGV) (Robinson et al. 2011) alignments from minimap2 assembly of targeted reads from FSHD1 participant P1 with a 4qA 5U contraction mapped to the 4qA D4Z4 region (upper panel) and a Chr 22, divergent DUX4 cluster (lower panel). LSAU-BSAT composite annotations are from the T2T-CHM13 RepeatMasker (http://www.repeatmasker.org) track and arrows indicate the location of the p13E-11/pLAM Cas9 cleavage sites.