Using Oxford Nanopore long-range sequencing to phase ABCA4

Abstract

Purpose : Mutations in ABCA4 are the most prevalent cause of monogenic retinal disease and are responsible for a range of phenotypes including Stargardt disease and cone-rod dystrophy. ABCA4-disease is caused by biallelic variants and recent studies have revealed pathogenic hypomorphic common variants and complex alleles (two or more variants in cis). The ability to cheaply and efficiently phase ABCA4 variants is therefore important to establish the biallelic status of variants when diagnosing singleton cases and to facilitate the further investigation of complex alleles. The goal of this study was to develop a cost-effective long-range sequencing method to phase ABCA4.

Methods : Long-range PCR primers encompassing the entire 130kb ABCA4 locus in overlapping blocks of 6-12kb were used to amplify genomic DNA using the Sequel Prep Long-Range PCR Kit. Samples were purified using AxyPrep Mag PCR Clean Up Kit and equimolar concentrations of each amplicon were combined and run on an Oxford Nanopore MinION with a flongle adaptor. Fast5 files were converted to Fastq files using Guppy, trimmed using Porechop and aligned using Minimap. Variants were called using Nanopolish and haplotypes assembled using WhatsHap.

Results : A pilot test involved amplifying 2 previously phased cases (eg. CEPH NA12878) and 2 Stargardt patients with two unphased variants. Initially 23 amplicons ranging between 6kb and 12kb in size and overlapping by a minimum of 2.5kb were used. One sample per flongle achieved an average read depth of ~7500, reducing stochastic effects. This method could be used to successfully phase the ABCA4 gene into 4 phase blocks which confirmed the phasing of previously identified variants in the control samples. The gaps were caused by a lack of variants in the overlap regions so amplicons were redesigned for these regions after analysing WGS datasets to ensure the capture of maximum variation.

Conclusions : This study has designed and tested a long-range, low-cost sequencing strategy to phase ABCA4. This will increase the accuracy of diagnosing individuals which is becoming increasingly important with the advent of therapies. It also has far reaching implications for the investigation into the contribution of complex alleles in ABCA4 related disease.

This is a 2021 ARVO Annual Meeting abstract.