A dual-mode targeted Nanopore sequencing assay for comprehensive SMN1 and SMN2 variant analysis

Abstract

BackgroundSpinal 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 homologous SMN1 and SMN2 genes. MethodsWe developed a dual-mode PCR-based target enrichment that generates 2.7 to 11.2 kb amplicons spanning SMN1 and SMN2 genes 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. ResultsWe 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 known SMN1 and SMN2 genotypes. 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. ConclusionWe 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 GenesSMN1 survival of motor neuron 1, telomeric HGNC:11117 SMN2 survival of motor neuron 2, centromeric HGNC:11118 CFTR CF transmembrane conductance regulator HGNC:1884