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

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 homologous SMN1 and SMN2 genes. Methods We 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. 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 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. 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. ### Competing Interest Statement BH, BK, and GL are employees of Bio-Techne with stock and stock options in this company. ### Funding Statement This study received funding support from Asuragen and Oxford Nanopore Technologies in the form of reagents and consumables. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Dubai Health Authority Research Ethics Committee gave ethical approval for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript