Parallel in-depth analysis of repeat expansions in ataxia patients by long-read sequencing

Abstract

Instability of simple DNA repeats has been known as a common cause of hereditary ataxias for over 20 years. Routine genetic diagnostics of these phenotypically similar diseases still rely on an iterative workflow for quantification of repeat units by PCR-based methods of limited precision. We established and validated clinical nanopore Cas9-targeted sequencing (Clin-CATS), an amplification-free method for simultaneous analysis of ten repeat loci associated with clinically overlapping hereditary ataxias. The method combines target enrichment by CRISPR/Cas9, Oxford Nanopore long-read sequencing, and a bioinformatics pipeline utilizing the tools STRIque and Megalodon for parallel detection of length, sequence, methylation, and composition of the repeat loci. Clin-CATS allowed for the precise and parallel analysis of 10 repeat loci associated with adult-onset ataxia and revealed additional parameter such as FMR1 promotor methylation and repeat sequence required for diagnosis at the same time. Using Clin-CATS we analyzed 100 clinical samples of undiagnosed ataxia patients and identified causative repeat expansions in 28 patients. Parallel repeat analysis enabled a molecular diagnosis of ataxias independent of preconceptions based on clinical presentation. Biallelic expansions within RFC1 were identified as the most frequent cause of ataxia. We characterized the RFC1 repeat composition of all patients and identified a novel repeat motif, AGGGG. Our results highlight the power of Clin-CATS as a readily expandable workflow for the in-depth analysis and diagnosis of phenotypically overlapping repeat expansion disorders.

Evaluation of Automated Magnetic Bead–Based DNA Extraction for Detection of Short Tandem Repeat Expansions With Nanopore Sequencing

“…Additionally, we determined the STR expansions in the genes FXN and C9ORF72 in extensive length distributions that probably reflects the somatic mosaicism of large repeat expansions due to the instability of the repeat. This has already been described for the investigated genes [26,27] and is also in accordance with the observation by Erdmann et al [8] for large expansions in the gene FMR1 using nanopore sequencing.…”

“…Its use was already demonstrated for the validation of new STR expansions that were initially identified in short-read sequencing data [5,6]. In order to characterize several known STR loci with targeted nanopore sequencing simultaneously, at least two different approaches have been developed [7,8]. First, Stevanovski et al [7] applied adaptive sampling to detect and analyze 37 STR expansion loci.…”

“…Adaptive sampling allows targeted enrichment using a real-time data analysis by recognition and acceptance or rejection of single DNA fragments through a programmable target selection [9,10]. Second, Erdmann et al [8] used a nanopore Cas9-targeted long-read sequencing approach for STR expansion detection.…”

“…Therefore, commercially available high-molecular-weight kits and several other DNA extraction protocols have been evaluated for specific applications [11,12]. For STR expansion detection with nanopore sequencing, Stevanowski et al [7] used two commercially available high-molecular-weight DNA extraction kits, a salting-out precipitation DNA purification and a silica-based DNA purification, whereas Erdmann et al [8] used a conventional automated magnetic bead-based DNA extraction method. In short, the detection of STR expansions requires DNA fragments of sufficient length whereas DNA integrity depends on the method of DNA isolation and on the preservation of the blood sample.…”

Exome Sequencing and Multigene Panel Testing in 1,411 Patients With Adult-Onset Neurologic Disorders

“…To detect STR expansions and indels in repeat regions (such as the OPRIs in PRNP), long read sequencing techniques are promising and will probably replace currently used time-consuming and labor-intensive strategies. 42,43 Another layer of molecular complexity in RNDs is the occurrence of pathogenic variants in the mtDNA. Exome capture kits in diagnostic settings generally do not include mtDNA.…”