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

Abstract

Background: Mutations in GBA cause Gaucher disease when biallelic and are strong risk factors for Parkinson's disease when heterozygous. GBA analysis is complicated by the nearby pseudogene. We aimed to design and validate a method for sequencing GBA using long reads.

Methods: We sequenced GBA on the Oxford Nanopore MinION as an 8.9 kb amplicon from 102 individuals, including patients with Parkinson's and Gaucher diseases. We used NanoOK for quality metrics, NGMLR to align data (after comparing with GraphMap), Nanopolish and Sniffles to call variants, and WhatsHap for phasing.

Results: We detected all known missense mutations in these samples, including the common p.N409S (N370S) and p.L483P (L444P) in multiple samples, and nine rarer ones, as well as a splicing and a truncating mutation, and intronic SNPs. We demonstrated the ability to phase mutations, confirm compound heterozygosity, and assign haplotypes. We also detected two known risk variants in some Parkinson's patients. Rare false positives were easily identified and filtered, with the Nanopolish quality score adjusted for the number of reads a very robust discriminator. In two individuals carrying a recombinant allele, we were able to detect and fully define it in one carrier, where it included a 55-base pair deletion, but not in another one, suggesting a limitation of the PCR enrichment method. Missense mutations were detected at the correct zygosity, except for the case where the RecNciI one was missed.

Conclusion: The Oxford Nanopore MinION can detect missense mutations and an exonic deletion in this difficult gene, with the added advantages of phasing and intronic analysis. It can be used as an efficient research tool, but additional work is required to exclude all recombinants.

Keywords: GBA; Gaucher disease; Oxford Nanopore MinION; Parkinson’s disease; long-read sequencing; mutation detection; mutation phasing.

Figure 1

Missense mutations detected with R9.4 chemistry. The IGV trace is shown for each sample with a mutation. The mutated base is shown, with 20 bases on either side. The three SNPs which comprise RecNciI are shown in Figure 3c. GenBank reference sequence NM_000157.3

Figure 2

Detection and phasing of a 55‐base pair exonic deletion in S5. The coverage track, with eight SNVs highlighted, and a selection of reads are shown, over exons 9 and 10 (chr1:155,204,981–155,205,661; NGMLR alignment). The deletion is clearly visible as a drop in coverage (red bracket). Reads are grouped and colored by haplotype for these variants, which are all on the blue‐colored reads. The arrows point to the SNVs (red = coding, blue = noncoding) and the red box to the deletion. GenBank reference sequence NM_000157.3

Figure 3

Evaluation of recombinant detection. A–C: IGV summary views over the region, including uncorrected allele frequencies at the three SNV positions. A: Nanopore sequencing does not detect even low levels of the three SNVs in bc74. B: Sample without RecNciI shown for comparison. C: The three RecNciI SNVs in this exon are clearly seen in sample S4 which carries RecTLdel55. D‐E: Sanger sequencing of exon 9–11 amplicon from bc74. D: When amplified directly from genomic DNA, RecNcil SNVs are seen (arrows). E: These are absent in nested PCR from GBA amplicon used for nanopore sequencing