Oxford Nanopore third generation sequencing for analysis of FMR1 5'UTR CGG repeat expansions.

Objective: Fragile X syndrome is mainly caused by the expansion of GC-rich cytosine-guanine-guanine (CGG) repeat in FMR1 5'UTR region, as well as rare gene point mutations or deletions in its open reading frame. Currently, third-generation long-read sequencing is a potential technology for simultaneously detecting CGG repeat expansions, point mutations, and deletions. However, a major challenge remains in obtaining the target long-fragment CGG repeat region with ultra-high GC content for sequencing.

Methods: We developed a novel approach combining long-fragment ultra-high GC polymerase chain reaction (PCR) amplification with Oxford Nanopore sequencing to detect the full spectrum of FMR1 5'UTR CGG repeat mutations. The method was validated using 10 standard cell line samples (males: n = 1, n = 1, n = 1, and n = 2; females: n = 1, n = 1, n = 2, and n = 1) and 53 retrospective clinical blood samples (males: n = 7, n = 3, n = 15, and n = 2; females: n = 9, n = 13, and n = 4).

Results: Our method demonstrated that the 100 % concordance with the triplet repeat-primed PCR and Southern blot analysis in genotyping 10 cell line samples and 53 clinical samples. Additionally, CGG repeat numbers showed strong correlation with reference mehods (male cell lines, n = 5, R = 0.9996; female cell lines, n = 5, R = 0.9972; clinical male samples, n = 26, R = 1.0000; clinical female samples, n = 25, R = 0.9854).

Conclusion: This study presents a simple and cost-effective strategy for preparing FMR1 5'UTR CGG repeat regions with long-fragment ultra-high GC content for third-generation sequencing. The approach could serve as a model for detecting other challenging disorders caused by short tandem repeat expansions, such as myotonic dystrophy and Huntington' s disease.