Whole exome sequencing-based copy number variant detection in inherited retinal disease

Abstract

Purpose : Whole exome sequencing (WES) is frequently used for molecular diagnostics of inherited retinal disease (IRD). WES allows the identification of both single nucleotide variants (SNVs) and copy number variations (CNVs). The purpose of this study was twofold: 1) to evaluate the performance of a novel WES enrichment kit - SureSelectXT Low Input Human All Exon V7 - for known IRD genes, and 2) to evaluate 4 different tools for CNV detection, and apply the best one on an IRD cohort in whom no causal genotype was identified following SNV analysis.

Methods : WES was performed using the SureSelectXT Human All Exon V6 kit (SSXT V6, 48 samples) and the recent SureSelectXT Low Input Human All Exon V7 kit (SSXT LI V7, 64 samples) (Agilent), followed by 2*150bp sequencing (HiSeq 3000, Illumina, 8 samples/lane). Four tools were used to compare CNV detection on a selection of 33 previously identified CNVs: cn.mops, Conifer, CNVkit and ExomeDepth. Novel CNVs were confirmed using qPCR.

Results : The SSXT LI V7 kit results in a higher % reads on-target (74%) compared to SSXT V6 (67%), as well as a higher median coverage of 272 known IRD genes (95x vs 76x) (RetNet). Comparison of 4 CNV detection tools identified ExomeDepth as the best one, able to call 29/33 known CNVs, followed by Conifer (20/33), CNVkit (16/33) and cn.mops (9/33). As a proof of concept, CNV detection on 18 IRD cases identified two heterozygous deletions, both confirmed by qPCR. The first one, identified in a sporadic retinitis pigmentosa (RP) case, encompasses part of the OSCAR gene and the NDUFA3, TFPT and PRPF31 genes. Overlapping deletions have previously been described in autosomal dominant RP with incomplete penetrance. The second deletion spans the last 3 exons of the RLBP1 gene and was found in a heterozygous state in a mother and her two sons, diagnosed with cone-rod dystrophy and myopia. An overlapping homozygous deletion is known to cause retinitis punctata albescens. No second RLBP1 SNV was found in the WES data of the family. CNV detection is ongoing in > 150 mutation-negative IRD cases following SNV analysis.

Conclusions : The SSXT LI V7 kit is a good alternative for the SSXT V6 kit, offering a higher median coverage, lower required input material and a shorter workflow. ExomeDepth was shown to perform best for CNV detection on WES samples, and allowed the identification of two heterozygous deletions in IRD.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.