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Analysing SNVs, SVs and phasing using targeted nanopore sequencing


Gaucher disease (GD), the most common lysosomal storage disorder, is caused by homozygous or biallelic mutations in the GBA gene. Heterozygous mutations in this gene are also a significant risk factor for Parkinson’s disease and other disorders1. The complex structure of the genomic region incorporating GBA, which includes multiple pseudogenes (with up to 96% homology), complicates analysis using PCR and traditional short-read DNA sequencing techniques. Longsequencing reads offer an alternative, more streamlined solution for complete analysis of the GBA gene. In order to assess this the validity of this approach, a team of researchers from the UK and USA utilised the MinION in combination with long-range PCR to amplify and sequence the entire ~8 kb gene2.

Long nanopore reads allowed more accurate characterisation of a 55 bp exonic deletion.

The team amplified the GBA gene from brain or saliva samples taken from different individuals with GD. All 10 samples were multiplexed and run on a single MinION flow cell, delivering 150- 500x coverage. Reads were aligned to a human reference genome (Hg19) using both the Graphmap and NGMLR tools prior to single nucleotide variant(SNV) calling using nanopolish. All previously characterised coding missense mutations were correctly identified. In addition, a number of non-coding SNVs were detected and any false-positives, while rare, could be easily identified and excluded. The team found that the NGMLR alignment tool provided optimal results and recommended a coverage of >300x for accurate determination of zygosity. Using the Sniffles and nanopolish tools for structural variant analysis, the team detected a single 55 bp exonic deletion in one of the samples.

The long reads provided by nanopore sequencing allowed more accurate characterisation of this SV than was possible using previous short-read based methodology – indicating a different site of recombination than previously thought. A further advantage of the long nanopore reads was the facility to phase the variants, which helps overcome the requirement to analyse relatives in clinical research samples. Using the Whatshap tool, the team were able to confirm compound heterozygosity in all relevant samples.

Commenting on this research, the team state: ‘The rapid evolution of specific bioinformatic methods, and the improvements in accuracy and data yield, combined with the minimal footprint and capital investment, make the MinION a suitable platform for long-read sequencing of difficult genes such as GBA, both in the diagnostic and research environments’ 2.*

The team now plan to amplify the whole genomic region incorporating the GBA gene and pseudogene (located 20 kb downstream) to fully characterise structural variation across the entire region of interest.

human fig 4.PNGFigure 1: Data analysis workflow. Only downstream analysis tools recommended by the authors are presented; however, other tools were assessed.2

* Nanopore devices are currently for research use only.

This case study is taken from the human white paper.

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1. Proukakis, C. Detection of GBA missense mutations and other variants using Oxford Nanopore MinION. Presentation. Available at: detection-gba-missense-mutations-andother-variants-using-oxford-nanopore-minion [Accessed:1 August 2019]

2. Leija-Salazar, M. et al. Detection of GBA missense mutations and other variants using the Oxford Nanopore MinION. BioRxiv 288068 (2018).

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