Cost-Effective Cas9-Mediated Targeted Sequencing of Spinocerebellar Ataxia Repeat Expansions

Hereditary repeat diseases are caused by an abnormal expansion of short tandem-repeats in the genome. Among them, Spinocerebellar ataxia (SCA) is a heterogenous disease, and currently, 16 responsible repeats are known. Genetic diagnosis is obtained by analyzing the number of repeats through separate testing of each repeat. Although simultaneous detection of candidate repeats using current massively parallel sequencing technologies has been developed to avoid complicated multiple experiments, these methods are generally expensive. In this study, we developed a cost-effective SCA repeat panel (FLO-SCAp) using Cas9-mediated targeted long-read sequencing and the smallest long-read sequencing apparatus, “Flongle.” This panel enabled the detection of repeat copy number changes, internal repeat sequences, and DNA methylation in seven patients with different repeat expansion diseases. The median (interquartile range) of coverage and on-target rate were 39.5 (12-72) and 11.6% (7.5-16.5), respectively. To validate our approach, we compared repeat copy number changes measured by FLO-SCAp and short-read whole-genome sequencing (SR-WGS). A high correlation was observed between FLO-SCAp and SR-WGS when the repeat length was 250 base pairs or less (r = 0.98, p <0.001). Thus, FLO-SCAp represents the most cost-effective method for conducting multiplex testing of repeats and can serve as the first-line diagnostic tool for SCA.