A Dual-Mode Targeted Nanopore Sequencing Assay for Comprehensive SMN1 and SMN2 Variant Analysis

Spinal muscular atrophy (SMA) is one of the most common recessive disorders, for which several life-saving treatment options are available. It is therefore essential to establish universal SMA screening and diagnostic programs using scalable, cost-effective, and accessible platforms to accurately identify all variation types. This task is complicated by high sequence homology between the SMN1 and SMN2 genes. Toward this goal, a dual-mode PCR-based target-enrichment method was developed, optimized, and evaluated in an external laboratory as a proof-of-concept for scalable and deployable any-length nanopore sequencing. The assay generates 2.7- to 11.2-kb amplicons spanning exons 3 to 8 of the SMN1 and SMN2 genes, which are then analyzed using a variant calling model that reports sequence and copy number variants specific to each gene from paralog-specific sequences and read-depth data. Overall, the assay detected single-nucleotide variants, insertions/deletions, and copy number variants with >98% genotype agreement across >750 samples, including cell lines, residual presumed-normal whole-blood donors, and patients with known SMN1 and SMN2 genotypes. The assay also demonstrated a dynamic sample throughput, 9-hour turnaround time, and 4-hour hands-on time. Together with the modest capital investment and consumable costs per sample, this assay can help to increase access to SMA testing in low- and middle-income settings. As a result, this PCR/Nanopore sequencing assay and analysis pipeline has the potential for universal implementation in SMA carrier screening and diagnostic programs.