Nanopore Long-Read Sequencing as a First-Tier Diagnostic Test to Detect Repeat Expansions in Neurological Disorders

Abstract

Inherited neurological disorders, such as spinocerebellar ataxia (SCA) and fragile X (FraX), are frequently caused by short tandem repeat (STR) expansions. The detection and assessment of STRs is important for diagnostics and prognosis. We tested the abilities of nanopore long-read sequencing (LRS) using a custom panel including the nine most common SCA-related genes and FraX and created raw data to report workflow. Using known STR lengths for 23 loci in 12 patients, a pipeline was validated to detect and report STR lengths. In addition, we assessed the capability to detect SNVs, indels, and the methylation status in the same test. For the 23 loci, 22 were concordant with known STR lengths, while for the last, one of three replicates differed, indicating an artefact. All positive control STRs were detected as likely pathogenic, with no additional findings after a visual assessment of repeat motifs. Out of 226 SNV and Indel variants, two were false positive and one false negative (accuracy 98.7%). In all FMR1 controls, a methylation status could be determined. In conclusion, LRS is suitable as a diagnostic workflow for STR analysis in neurological disorders and can be generalized to other diseases. The addition of SNV/Indel and methylation detection promises to allow for a one-test-fits-all workflow.

Keywords: Oxford Nanopore Technologies; SNV; fragile X syndrome; indel; long read sequencing; methylation; neurological disorders; short tandem repeat; spinocerebellar ataxia.

Figure 1

MOLGENIS VIP report for a fictitious sample. The report consists of two sections. (a) Upon opening the report, the first section shows an overview of all the STRs in the panel, along with allele repeat lengths, the suggested classification, and the number of reads supporting the call (coverage). The block at the left allows the user to specify that only STRs with a specific classification (e.g., VUS, LP, and P) are shown, which in this example would mean only the ATXN3 repeat is shown. For each variant, the most important metrics are shown, including the repeat confidence interval (CI), coverage, and reads spanning the entire repeat for both alleles. (b) The second section can be opened by clicking on a specific variant. It provides detailed information for this variant. The top section is an IGV view of the repeat region. The bottom section gives all available annotations for this variant. The example shows the variant view for the ATXN3 repeat.

Figure 2

Methylated (red) and unmethylated (blue) FMR1 promoter regions. For each sample, the region depicted is chrX:147,911,892-147,912,180. (a) Control XY, no repeat expansion; (b) control XX, no repeat expansion, (c) affected male, XY, mosaic repeat expansion, and (d) female carrier, XX, repeat expansion.