Nanopore Ultra-Long Sequencing for FSHD: From Molecular Diagnosis to Preimplantation Genetic Testing

Figures

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  Figure 1. Pathogenic mechanisms and clinical subtypes of FSHD.

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  Figure 2. Nanopore sequencing core mechanism and technological development timeline. (A) Schematic representation of the Nanopore sequencing principle: single-stranded DNA translocates through a protein Nanopore embedded in a synthetic polymer membrane under an applied voltage, with a motor protein controlling translocation speed. (B) Key developmental milestones in ONT technology from the MinION early access program to R11 chemistry. (C) Hardware platform comparison: from portable MinION for point-of-care use to high-throughput PromethION P48 for population-scale whole-genome sequencing.

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  Figure 3. Nanopore ultra-long read sequencing for FSHD diagnosis workflow. (A) Classify reads originating from chr4 or chr10, and filter ultra-long reads that fully span the D4Z4 repeat array. (B) Detect pLAM haplotype markers and enumerate D4Z4 repeat units in each spanning read, annotating each read with chromosome, haplotype, and repeat copy number. (C) Cluster spanning reads by chromosome and haplotype to resolve complete D4Z4 arrays and obtain precise repeat counts for each haplotype.

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  Figure 4. Nanopore ultra-long read sequencing for the FSHD PGT-M workflow. (A) FSHD diagnostic workflow based on Nanopore ultra-long read sequencing: including UHMW DNA library preparation, ultra-long read sequencing, and single-molecule resolution of D4Z4 repeat counts and 4qA haplotypes. (B) Haplotype phasing of D4Z4-flanking variants using the T2T-CHM13 reference genome to distinguish disease-linked and normal allelic backgrounds. (C) Application of phased haplotype markers in FSHD preimplantation genetic testing, for pathogenic haplotype tracing and embryonic risk stratification.