The Journal of Molecular Diagnostics: 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 lifesaving 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.

Authors:
Bradley Hall,* Sawsan Alyafei,† Sathishkumar Ramaswamy,† Shruti Sinha,† Maha El Naofal,† Fatima Rabea,† Bryan J. Killinger,* Gary J. Latham,* and Ahmad Abou Tayouny†§

From Asuragen,* A Bio-Techne Brand, Austin, Texas; and the Dubai Health Genomic Medicine Center,† the Mohammed Bin Rashid University of Medicine and Health Sciences,‡ and the Center for Genomic Discovery,§ Mohammed Bin Rashid University of Medicine and Health Sciences, Dubai Health, Dubai, United Arab Emirates