Comparison of CytoSure Constitutional NGS with microarrays for CNV detection

Introduction

For the detection of copy number variation (CNV), DNA microarrays are viewed as the ‘gold standard’. The use of array comparative genomic hybridisation (aCGH) has been particularly effective in detecting CNVs within DNA samples from individuals with intellectual disability (ID) and developmental delay (DD). This has resulted in the detection of novel syndromes which were previously undetectable¹. In order to further facilitate cytogenetics research, specific microarrays, such as the CytoSure^® Constitutional v3 array, have been developed. These microarrays have an enhanced probe coverage for genes of interest as well as probes regularly spaced throughout the genome to detect larger CNVs on the genomic backbone.

Next Generation Sequencing (NGS) has become a revolutionary technology for the analysis of ID and DD samples, having the advantage of being able to detect single nucleotide variations (SNVs) and small insertions and deletions (indels) simultaneously within many genes. However, the ability of NGS to call small CNVs is still not routine and robust. In order to facilitate the transition of small (as low as single exon) CNV calling to NGS from arrays, appropriate capture panel design and improved software are both required.

OGT, with its world-leading probe design and software capabilities, is well placed to meet this need. We have designed an NGS assay and analysis software that is able to detect large and small CNVs with the same precision and sensitivity as microarrays, as well as providing SNV and Indel calling.

The CytoSure Constitutional NGS assay is a hybridisation-based method to capture specific regions of the genome. These regions are:

• Over 700 genes implicated in ID and DD at the single exon level – to detect SNV/Indels within the genes and UTRs as well as small intragenic CNVs.
• Nucleotides flanking target genes, up to 35 base pairs from the exons to capture splice site variants.
• Extensive backbone baits spaced throughout the genome – to detect large CNVs and stretches of loss of heterozygosity (LOH).

Like OGT’s CytoSure Constitutional arrays, the gene list has been developed with input from the ClinGen database and the Deciphering Developmental Disorders project, as well as leading Cytogeneticists. In addition to the targeted gene/exon regions, there are 28,641 backbone baits which are spaced according to the priority regions in the genome and provide an estimated CNV resolution of 189kb in high priority areas and a LOH resolution of 5Mb in the non-targeted region.

This technical note compares performance of CytoSure Constitutional NGS and a range of microarrays for 255 research samples processed in three independent laboratories and OGT.