Long-read sequencing: bridging the diagnostic gap for undiagnosed cases in Chile

Abstract Hereditary ataxias, like other rare diseases, pose a significant diagnostic challenge due to their heterogeneity and genetic complexity, which includes repeat expansions, single nucleotide variants, insertions, deletions, and duplications. Despite extensive phenotypic characterization and numerous developed panels and protocols, the global diagnostic yield is still only around 50%, which is often constrained by economic barriers. Evidently, at our center in Santiago, Chile, the diagnostic yield was 23% from 2018 to 2022 using the standard whole-exome sequencing approach. Since then, we have recruited 50 patients with unresolved hereditary cerebellar ataxias. A single test detecting all genetic variation causing ataxia is ideal, hence long-read sequencing offers a more comprehensive solution. To bridge the gap in undiagnosed cases with suspected monogenic disorders, we developed a low-cost extraction method for long-read sequencing, freely available at dx.doi.org/10.17504/protocols.io.n92ldmx3ol5b/v1, which is now used across low-income sites within the Global Parkinson’s Genetics Program. For our research, we collected 25 samples and prioritized 14 cases, including four ataxic and 10 Parkinsonism patients (two atypical cases and eight familial with negative results from previous genetic studies). Applying this method, we generated high-quality nanopore whole-genome sequencing data, with an average N50 of 21 kb and 130 Gb per flow cell. Further, using the Napu tool, we identified disease-associated variants and assessed DNA methylation at known ataxia loci, efficiently resolving the genetic architecture in many of these cases. Our findings highlight the utility of long-read sequencing in elucidating the genetic basis of complex neurological disorders. Biography Paula Saffie is a Chilean MD, pursuing a PhD in Brazil with a Michael J. Fox Foundation grant to study the genetics of Parkinson's disease in Chile and southern Brazil. In 2016, we diagnosed the first ARSACS case in Chile after the patient had sought a diagnosis for 16 years, which motivated her to study undiagnosed cases. After a diagnostic odyssey for a family from southern Chile, we reported the first FGF14 expansions in Latin America. During her PhD, Paula has learned skills from DNA extraction to programming; and now aims to gain training and experience in long-read sequencing to potentially shorten the diagnostic journey for many patients.