Using Oxford Nanopore technology to detect triplet expansion in Spinocerebellar Ataxia 7

Abstract

Spinocerebellar ataxia type 7 (SCA7), an autosomal dominant disease, is caused by the expansion of the CAG repeat sequence in the ATXN7 gene. The current diagnostic test for SCA7 is by capillary electrophoresis which involves size characterization of the CAG repeat expansion. This provides a good estimate of the number of CAG repeats but gives no information on the sequence variation within the expansion. The aim of the project was to assess the effectiveness of using Oxford Nanopore long-range single molecule sequencing to characterize the length and sequence of the triplet repeat tract in the ATXN7 gene. The first step was primer design and PCR optimization to develop a robust amplification protocol to ensure that African DNA samples would amplify, by minimising PCR failure due to variations in primer binding sites. The PCR optimization was designed for long stretches of G-C rich regions to accommodate the maximum size of a SCA7 triplet expansion. Ten control DNA samples and two patient samples were used in this project and following PCR, they were sequenced using the Oxford Nanopore MinION device. Data analysis protocols recommended by the manufacturer could not be used because the size of the reads was too short for the standard algorithm (<1kb). Alternative data analysis methods were used, which analysed the triplet number from the read size, but the analysis of the sequence remained suboptimal. A cost analysis was attempted to compare the costs of conducting a size analysis by capillary analysis against using nanopore sequencing, but had many limitations. Future recommendations are to design primers that capture a longer fragment of the ATXN7 gene to produce longer reads, as used in other successful, recently published analyses of nanopore data.