A dual-mode targeted Nanopore sequencing assay for comprehensiveSMN1andSMN2variant analysis

Abstract

Background Spinal Muscular Atrophy (SMA) is one of the most common recessive disorders for which several life-saving treatment options are currently available. It is essential to establish universal SMA screening and diagnostic programs using scalable, cost-effective and accessible platforms to accurately identify all variation types, which is complicated by homologousSMN1andSMN2genes. Methods We developed a dual-mode PCR-based target enrichment that generates 2.7 to 11.2 kb amplicons spanningSMN1andSMN2genes for any-length nanopore sequencing. We trained a variant calling model that utilizes paralog-specific sequences and read-depth data to accurately detect sequence and copy number variants specific to each gene. Results We present results from the development, optimization, and external evaluation of this assay using over 750 samples, including cell lines, residual presumed normal blood donors, and patients with knownSMN1andSMN2genotypes. The assay detects SNVs, indels, and CNVs with >98% accuracy across all sample sets, with a highly dynamic throughput range, relatively fast turnaround time, and limited hands-on-time. Together with the modest capital investment and consumable costs per sample, this assay can help increase access to SMA testing in low- and middle-income settings. Conclusion We describe a PCR/Nanopore sequencing assay and a customized analysis pipeline for the comprehensive and accurate detection of variation at the SMA locus and demonstrate its scalability, cost-effectiveness, and potential for the universal implementation of SMA screening and diagnostic programs. Human Genes SMN1survival of motor neuron 1, telomeric HGNC:11117SMN2survival of motor neuron 2, centromeric HGNC:11118CFTRCF transmembrane conductance regulator HGNC:1884