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

Figures

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  Figure 1.. Cas9-targeted nanopore sequencing of D4Z4 repeats. (A) Schematic of D4Z4 repeat arrays found in sub-telomeric regions of human chr4q and 10q. A 6U D4Z4 array is shown on the permissive 4qA haplotype, and blue/red shading of repeats indicates CpG methylation. (B) Locations of Cas9-guide RNA cleavage sites at the centromeric (p13E-11) end, telomeric (pLAM) end, and within each repeat (D4Z4). 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, non-overlapping windows). Colors indicate reads mapped to the chr4q (blue) and 10q (green) D4Z4 arrays, as well as divergent DUX4 clusters (purple). (D) IGV browser alignments of targeted reads from FSHD1 participant P5 with a 4qA 6U contraction. The D4Z4/BlnI/XapI ratio shows the number of 3.3 kb D4Z4 repeats contained with the aligned reads and the number of 10q-specific (BlnI) and 4q-specific (XapI) restriction sites found within those repeats. The lower panel shows alignments to a divergent D4Z4 repeat cluster on chr22.

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  Figure 2.. CpG methylation gradients traverse chr4q and 10q D4Z4 arrays. (A) Methylation levels of p13E-11 to pLAM targeted reads summarized as % 5mC sites per D4Z4 repeat (3.3 kb KpnI-to-KpnI region with ~320 CpG sites per repeat). Repeat numbering begins at the centromeric end (p13E-11) and is reversed for the chr4 pLAM (telomeric) reads. (B) Single-read plots of targeted reads from (A) using modbamtools with unmethylated CpGs represented in blue and 5-methyl CpGs in red. The schematic representation of D4Z4 repeats shows DUX4 exons 1 and 2 and the CTCF insulator region, and the green stripe indicates the 10th centromeric D4Z4 repeat. (C) Alignments and methylation plots of untargeted reads (R10.4.1_e8.2 nanopores) from a control subject (C4) mapped to the 4qB 16U haplotype, the 4qA 43U haplotype, the 10q 20U haplotype, and the 10q 21U haplotype. The grey stripe delineates the start of the syntenic telomeric region shared between 4q and 10q, and the green stripe marks the 10th repeat.

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  Figure 3.. Methylation gradient modeling. (A) Intercept and slope estimates from p13E-11 to pLAM 4qA 6U reads from participant P1. The red line is the model estimate and the thin lines are the individual read estimates with a color scale for read-level total % 5mC. The right panel shows the observed D4Z4 methylation frequencies for each read (points) with their individual read estimates. The 68 plots are ordered by read-level % 5mC. (B) Intercept and slope estimates as in (A) from FSHD1, FSHD2 participants, and control subject C4 (see Table 2). (C) Model for gradient formation via basal methylation followed by asymmetric spreading. (D) Predicted D4Z4 methylation levels from simulated basal methylation and spreading (red points) compared to observed values (blue points, mean methylation per repeat, data from (B)). In the lower panel, the basal methylation and spreading parameters were reduced by a factor of 2.

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  Figure 4.. Fine-scale D4Z4 repeat methylation patterns. (A) Methylation plot of 4qA 6U reads from participant P1 using the “smooth_ksmooth” function (smoothness = 10) with single-read plots from modbamtools. (B) Annotated D4Z4 features including DUX4 exons 1 and 2 (mRNA from AF117653.3), the CTCF insulator (Blp1-Blp1 fragment from Ottaviani et al.,2010), BsaAI and FseI methylation-sensitive restriction sites, and PCR amplicons commonly used for bisulfite sequencing. A methylartist plot shows 322 CpG residues represented as blue (unmethylated) or red (methylated) dots for the R1 and R5 repeats from a subset of reads in (A). The middle panel shows the average individual CpG methylation from the 4qA 6U reads in (A) for the 322 CpGs, aligned from R1 to R5. The lower panel shows the methylation frequency plot smoothed as in (A), with the methylation range of six CpGs in the DUX4 proximal promoter with the transcription start from Dixit et al. (AF117653.3). (C) Methylation levels of individual 3.3 kb D4Z4-cut reads with % 5mC per read on the y-axis. The number of reads and mean/median % 5mC per read are shown for each chromosome. Upper plot = FSHD1 trio (affected father, affected daughter,…

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  Figure 5.. Methylation profiles of divergent D4Z4 repeats. (A) Annotated structure and copy number of the distal 4 kb D4Z4 repeat beginning at the 5’ KpnI site through the 4qAS pLAM sequence/γ-satellite junction (see Supplemental Table S2 for list of genomic locations). (B) Methylation profile of a full-length, divergent D4Z4 repeat found on chr 22. The LASTZ percent identity (PIP) plot shows the distribution of sequence identity along the length of this chr22 D4Z4 repeat compared to the distal chr 4qAS D4Z4 repeat. (C) Methylation plots of untargeted reads mapping to chr22:9,317,908–9,717,807 region from the C4 control (R10.4.1_e8.2 nanopores). (D) Distributions of CpG methylation levels of divergent D4Z4 repeats where each point represents the % 5mC of an individual read within the region that aligns completely with a divergent repeat. The C3 sample was sequenced using R10.4.1_e8.2 nanopores.

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  Figure 6.. Methylation gradients in other CpG-dense repeat regions. (A) Manhattan plot of CpG density in 33 kb sliding windows (step size = 3.3 kb) across the T2T CHM13 v2.0 reference genome. Windows above 7% CpG density are marked as green dots. Alignment and methylation plots of untargeted reads from the C4 control (R10.4.1_e8.2 nanopores) mapped to (B) the tRNA cluster on chromosome 1 and (C) the 5S rRNA cluster on chromosome 1. Mapped reads are anchored by unique flanking sequence and the two ends of the 5S rRNA cluster are shown separately. The annotations are the RepeatMasker repetitive element UCSC browser track.