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Emma Shipstone, James Reid, Sandra Kachhia, John Shovelton, Duarte Molha, Christina Taylor, Jagath Kasturiarachchi, Jolyon Holdstock, Venu Pullabhatla, Laura Parkes, Natalie Milner, Douglas Hurd



Array comparative genomic hybridisation (aCGH) has been used extensively in research to determine the causative copy number variation (CNV) in Intellectual Disability (ID) and Development Delay (DD) cases1. Microarrays have a limitation in that they cannot detect single nucleotide variations (SNVs), which may be causative. Conversely, robust small CNV calling via other genetic techniques, e.g. exome sequencing has remained challenging. To overcome these issues, we have designed an NGS assay and supporting analytical software that reliably detects a wide range of aberration types, including:

  • SNVs within 700 targeted ID and DD genes, exons and UTRs
  • Small (single exon) CNVs within the targeted 700 ID and DD genes
  • Larger CNVs (>190kb) across the genome
  • Loss of Heterozygosity regions with a resolution of <5Mb

The 700 targeted genes known to be important in ID and DD were carefully selected on the basis of ClinGen2 and DDD3 guidelines. The ability to detect these 4 different types of aberration significantly improves the likelihood of detecting causative aberrations compared to aCGH alone.

Outlined here is the design and testing of the assay using research samples obtained from cytogenetic laboratories. A particular focus of the testing has been confirmation that the CNV detection capabilities of the assay are as good as those obtained with aCGH (Figures 1-7). This has included testing with mosaic samples. We have demonstrated excellent concordance between the results obtained with the new assay and aCGH.





Small CNV

Figure 1: ~80kb duplication detected on chromosome 2Figure 1: [A] ~80kb duplication detected on chromosome 2 with both CytoSure® Constitutional NGS and [B] CytoSure Constitutional v3 4x180k array.

Figure 2: Single exon deletion in a female DMD sampleFigure 2: [A] Single exon deletion in a female DMD sample detected with CytoSure NGS and [B] CytoSure Constitutional v3 4x180k array.


Large CNV

Figure 3: 753kb deletion detected on chromosome 8Figure 3: [A] 753kb deletion detected on chromosome 8 on CytoSure Constitutional NGS and [B] on a 4x180k CytoSure Constitutional v3 array CGH.

Figure 4: 1.4Mb duplication on chromosome 17Figure 4: [A] 1.4Mb duplication on chromosome 17 shown with the Interpret software on CytoSure Constitutional NGS. [B] The matching array result with CytoSure Interpret software using a CytoSure 4x180k v2 ISCA array.



Figure 5: 8.8Mb stretch of LOH on chromosome 7 with a 213kb duplicationFigure 5: [A] 8.8Mb stretch of LOH on chromosome 7 with a 213kb duplication on CytoSure Constitutional NGS. [B] Equivalent 4x180k CytoSure Constitutional custom array.


Results II


Figure 6: Detection of an indel within the NDP gene on chromosome XFigure 6: Detection of an indel within the NDP gene on chromosome X.



Figure 7: Detection of an SNV in the MYBPC3 geneFigure 7: Detection of an SNV in the MYBPC3 gene.


Overall results

Figure 8: Summary of results from overall studyFigure 8: Summary of results from overall study.



We have demonstrated that CytoSure Constitutional NGS was able to detect CNVs with excellent concordance to microarrays, but has the added benefit of SNV/Indel and LOH calling. We were able to call all the CNVs from our research sample trial at an independent lab providing confidence in the NGS solution. We were also able to call the SNV aberrations from our research samples, providing a rich new source of genetic information in comparison to microarray alone.


  1. Sharp et al, Nature Genetics 38 p1038


  • Centre hospitalier universitaire de Sherbrooke, Quebec, Canada
  • Clinical Genome Resource (ClinGen)
  • Prof. Matthew Hurles, FMedSci, FRS, Wellcome Sanger Institute


CytoSure®: For Research Use Only; Not for Diagnostic Procedures. 

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