Performance Evaluation of a PCR/Nanopore Assay for Carrier Screening for Cystic Fibrosis, Spinal Muscular Atrophy,...

Cystic fibrosis, spinal muscular atrophy, and fragile X syndrome are among the most common inherited genetic disorders, making carrier screening essential for identifying at-risk couples. Traditional screening often involves multiple workflows and may miss rare variants. Comprehensive sequencing offers broader variant detection across diverse populations. This study validated a PCR/Nanopore-based assay for comprehensive assessment of CFTR, SMN1/2, and FMR1. Samples included anonymized DNA from: whole blood (archival clinical samples; n = 53), cell lines (n = 19), and residual College of American Pathology proficiency testing material (n = 3). Using the AmplideX Nanopore Carrier Plus reagents, gene-specific PCR was performed, followed by barcoding and sequencing on MinION flow cells. Data were analyzed using the AmplideX One Reporter software. Results for SMN1/2 and FMR1 showed 100% concordance with orthogonal methods (PCR/fragment analysis laboratory-developed tests) and 97% for CFTR. These results were reproducible between interrun and intrarun repeats. Here, it is shown that the PCR/Nanopore-based assay is accurate and reproducible for identifying pathogenic variants and poly-T size in CFTR; SMN1/2 copy number and single-nucleotide polymorphism detection; and FMR1 CGG repeat sizes and AGG interruptions. The assay was also able to detect mosaicism as low as 10% for FMR1. This single workflow enables accurate, reproducible screening for multiple disorders, with the ability to identify more CFTR variants than traditional genotyping panels.