Characterization of FMR1 Repeat Expansion and Intragenic Variants by Indirect Sequence Capture

Area of Science:

• Genetics
• Molecular Biology
• Bioinformatics

Background:

• Traditional methods for analyzing repeat expansions in genetic disorders like fragile X syndrome (FXS) lack single-nucleotide resolution and cannot characterize variants outside repeat regions.
• Next-generation sequencing offers potential but requires target enrichment for clinical application, with no current methods enabling parallel analysis of long DNA fragments using both long-read and short-read technologies.

Purpose of the Study:

• To investigate the use of indirect sequence capture (Xdrop technology) combined with Nanopore and Illumina sequencing for the parallel analysis of repeat expansions and single-nucleotide variants (SNVs)/indels in the FMR1 gene.
• To assess the efficiency and resolution of this combined approach for characterizing causative variants in FXS.

Main Methods:

• Utilized Xdrop technology for efficient enrichment (>200×) of large target DNA fragments (~60-80 kbp) encompassing the entire FMR1 gene.
• Performed Nanopore long-read sequencing for characterization of repeat lengths and interruptions.
• Conducted Illumina short-read sequencing for detection of SNVs and indels within the FMR1 gene.

Main Results:

• Achieved efficient enrichment of large FMR1 gene fragments.
• Nanopore sequencing enabled complete characterization of repeat lengths (>1 kbp) and interruptions in samples with normal, pre-mutation, and full mutation statuses.
• Illumina sequencing successfully detected SNVs and indels, identifying pathogenic variants outside typical repeat expansions.
• Demonstrated parallel analysis of repeat expansions and SNVs/indels at single-nucleotide resolution.

Conclusions:

• The combined Xdrop enrichment with Nanopore and Illumina sequencing provides a comprehensive approach for analyzing the FMR1 gene.
• This method allows for precise characterization of repeat expansions and other variants, offering single-nucleotide resolution.
• The system has the potential to improve FXS diagnosis, genetic counseling, and genotype-phenotype correlation studies with clinical optimization.