Bridging the gap: a prospective trial comparing programmable targeted long-read sequencing and short-read genome sequencing for genetic diagnosis of cerebellar ataxia

The cerebellar ataxias (CA) are a heterogeneous group of disorders characterized by progressive incoordination. Seventeen repeat expansion (RE) loci have been identified as the primary genetic cause and account for >80% of genetic diagnoses. Despite this, diagnostic testing is limited and inefficient, often utilizing single gene assays. This study evaluated the effectiveness of long- and short-read sequencing as diagnostic tools for CA. We recruited 110 individuals (48 females, 62 males) with a clinical diagnosis of CA. Short-read genome sequencing (SR-GS) was performed to identify pathogenic RE and also non-RE variants in 356 genes associated with CA. Independently, long-read sequencing with adaptive sampling (LR-AS) and performed to identify pathogenic RE. SR-GS identified pathogenic variants in 38% of the cohort (40/110). RE caused disease in 33 individuals, with the most common condition being SCA27B (n=24). In comparison, LR-AS identified pathogenic RE in 29 individuals. RE identification for the two methods was concordant apart from four SCA27B cases not detected by LR-AS due to low read depth. For both technologies manual review of the RE alignment enhanced diagnostic outcomes. Orthogonal testing for SCA27B revealed a 16% and 0% false positive rate for SR-GS and LR-AS respectively. In conclusion, both technologies are powerful screening tools for CA. SR-GS is a mature technology currently utilized by diagnostic providers, requiring only minor changes in bioinformatic workflows to enable CA diagnostics. LR-AS offers considerable advantages in the context of RE detection and characterization but requires optimization prior to clinical implementation.