ABCA4 variant phasing and complete haplo-genotype analysis for singletons using Cas9 targeted ultra-long read nanopore sequencing

Abstract

Purpose : Short, paired-end sequencing (next generation sequencing, NGS) enables interrogation of up to ~95% of the human genome but short reads lack genomic context: Importantly, variants greater than approximately 600bp apart cannot be phased. Thus, singleton patients are often left with inconclusive genetic findings. We previously showed results of Cas9 targeting of chromosomal segments (CATCH) with Oxford Nanopore Technologies (ONT) long-read sequencing for detection of a complex structural variant in ABCA4 and here we sought to investigate distant variant phasing in ABCA4 in two patients with suspected ABCA4-retinopathy.

Methods : Freshly isolated leukocyte nuclei were lysed and CATCH was performed for ABCA4 using CRISPR guides flanking the gene (145kb) followed by separation with 2-D electrophoresis using the SageHLS system (SAGE science). ONT MinION long-read sequencing was performed on resultant chromosomal fragments enriched forABCA4. Reads were basecalled (Guppy) and aligned to the human genome build 38 (GRCh38, Minimap2). Variant calling and haplotype data were generated using Clair3 and WhatsHap respectively.

Results : Two patients with Stargardt disease were unresolved following WGS. Patient 1 carried two heterozygous variants (ABCA4 c.1335C>G p.Ser445Arg, c.4256T>C p.Met1419Thr). Patient 2 harboured ABCA4 c.5882G>A p.Gly1961Glu and three candidate intronic variants (c.443-669G>A, c.1938-289G>T and c.5196+415T>C). No family members were available for segregation. CATCH-nanopore sequencing generated up to 99 on-target reads (chr1:93,992,834-94,121,148) with an average 33.6x read depth across the gene and N50 of 82kb with 10 full-length reads (patient 2). Variant calling and haplotyping demonstrated the phase of variant calls spanning the complete gene showing the two missense variants to be in trans for patient 1. Only c.1938-289G>T was in trans with c.5882G>A in patient 2. The deep intronic variant is predicted to introduce a strong donor site deep in intron 13 of the gene.

Conclusions : This study demonstrates that CATCH-nanopore sequencing can generate complete haplo-genotypes for ABCA4 in singleton patients unresolved by WGS. Up to 39x read depth was achieved and approximately 10% of reads were full length. This means that complete haplotypes can potentially be resolved for any ABCA4 variant of interest.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.