Characterization of FMR1 repeat-expansion and intragenic variants by indirect sequence capture

In this study, we explored the use of the Indirect Sequence Capture (Xdrop technology) coupled to Nanopore and Illumina sequencing to characterize FMR1, the gene responsible of Fragile X syndrome. From a single detection sequence of 100 bp, the approach yielded efficient enrichment (>200X) of large target-DNA-fragments (40 kbp) comprising the whole FMR1 gene. Analysis of Xdrop-enriched samples with Nanopore long-read sequencing allowed to properly characterize repeat lengths in a set of samples featuring normal, pre-mutation or full-mutation status (>1000 bp), as well as to correctly identify repeat interruptions, relevant for disease transmission and prognosis. From the same samples, Single-Nucleotide-Variants (SNV) and small Insertions and Deletions (InDels) could be analyzed by employing Illumina sequencing. This allowed to investigate the presence of possible pathogenic variants even within the FMR1 gene body, an option desirable to provide complete genetic counselling when the repeat characterization is inconclusive.