Fragile X Syndrome Carrier Screening Using a Nanopore Sequencing Assay

Fragile X syndrome (FXS) is the leading cause of monogenic autism spectrum disorder and inherited intellectual disabilities. Although the value of population-based FXS carrier screening has been acknowledged, appropriate screening methods are urgently required to establish and implement screening programs. We developed a nanopore sequencing-based assay that includes data analysis software to identify FXS carriers. Reference and clinical samples were used to evaluate the performance of the nanopore sequencing assay. Triplet-primed PCR and PacBio sequencing assays were used for comparisons. Nanopore sequencing identified reference carrier samples with a full range of premutation alleles in single-, 10-, and 100-plex assays, and identified AGG interruptions in an allele-specific manner. Moreover, nanopore sequencing revealed no size preference for amplicons containing different-length CGG repeat regions. Finally, nanopore sequencing successfully identified three carriers among 10 clinical samples for preliminary clinical validation. The observed variation in CGG repeat region size resulted from the base calling process of nanopore sequencing. In conclusion, the nanopore sequencing assay is rapid, high-capacity, inexpensive, and easy to perform, thus providing a promising tool and paving the way for population-based FXS carrier screening.