Detection of GBA missense mutations and other variants using the Oxford Nanopore MinION

Purpose: Mutations in GBA cause Gaucher disease when biallelic, and are strong risk factors for Parkinson9s disease when heterozygous. GBA analysis is complicated by the nearby pseudogene. We aimed to design and validate a method for sequencing GBA on the Oxford Nanopore MinION. Methods: We sequenced an 8.9 kb amplicon from DNA samples of 17 individuals, including patients with Parkinson9s and Gaucher disease, on older and current (R9.4) flow cells. These included samples with known mutations, assessed in a blinded fashion on the R9.4 data. We used NanoOK for quality metrics, two different aligners (Graphmap and NGMLR), Nanopolish and Sniffles to call variants, and Whatshap for phasing. Results: We detected all known mutations, including the common p.N409S (N370S) and p.L483P (L444P), and three rarer ones, at the correct zygosity, as well as intronic SNPs. In a sample with the complex RecNciI allele, we detected an additional coding mutation, and a 55-base pair deletion. We confirmed compound heterozygosity where relevant. False positives were easily identified. Conclusion: The Oxford Nanopore MinION can detect missense mutations and an exonic deletion in this difficult gene, with the added advantage of phasing and intronic analysis. It can be used as an efficient diagnostic tool.