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

Area of Science:

• Genomics
• Molecular Diagnostics
• Neurology

Background:

• Inherited neurological disorders like spinocerebellar ataxia (SCA) and fragile X (FraX) are often caused by short tandem repeat (STR) expansions.
• Accurate detection and assessment of STRs are crucial for diagnosing and predicting the prognosis of these conditions.

Purpose of the Study:

• To evaluate the utility of nanopore long-read sequencing (LRS) for detecting STR expansions in neurological disorders.
• To develop and validate a workflow for STR analysis using LRS.
• To assess the capability of LRS to simultaneously detect single nucleotide variants (SNVs), indels, and methylation status.

Main Methods:

• Utilized a custom nanopore LRS panel targeting nine common SCA-related genes and the FMR1 gene (for FraX).
• Validated a bioinformatics pipeline using known STR lengths from 23 loci in 12 patients.
• Assessed the detection of SNVs, indels, and methylation status in parallel with STR analysis.

Main Results:

• The LRS workflow demonstrated high concordance (22/23 loci) with known STR lengths, with one locus showing an artefact in replicates.
• Accurate detection of pathogenic STRs was achieved, with no additional findings upon visual inspection.
• The method achieved 98.7% accuracy for SNV and indel detection (2 false positives, 1 false negative out of 226 variants).
• Methylation status was successfully determined for FMR1 controls.

Conclusions:

• Nanopore long-read sequencing (LRS) is a suitable technology for a diagnostic workflow analyzing STRs in neurological disorders.
• The integrated approach allows for simultaneous detection of STRs, SNVs, indels, and methylation, paving the way for a unified diagnostic test.
• This LRS-based workflow has the potential to be generalized for the diagnosis of other genetic diseases.