A next generation sequencing solution to detect copy number variants, single nucleotide variants and loss of heterozygosity in intellectual disability and developmental delay samples

Wednesday 3 June 2020
Product types:

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

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The detection of Copy Number Variants (CNVs) in intellectual disability and developmental delay (ID/DD) samples is crucial in elucidating the genetic cause of abnormality. We have developed a targeted NGS panel and analytical software (Interpret) to accurately detect CNVs, as well as SNVs, indels and LOH.

The assay uses a bait capture approach, which is able to capture the exons and untranslated regions (UTRs) from over 700 genes, chosen for their relevance in ID/DD, as well as a range of backbone regions across the genome. Combined with OGTs proprietary CNV detection algorithm in the software, both intragenic and large ‘backbone’ CNVs can be detected robustly.

We implemented a web-based solution that runs OGTs NGS analysis pipeline, comprising many state-of-the-art open-source NGS software tools. These tools were carefully chosen and deployed using containers to ensure cross-platform compatibility and reproducibility. Pipeline optimisation and performance was assessed using equivalent array data and reference materials.

We will outline the results from over 200 intellectual disability and developmental delay research samples to demonstrate the efficiency of the CNV, SNV and LOH detection. The study demonstrated that the assay automatically called 100% of SNVs and 97% of reported pathogenic CNVs (including small intragenic CNVs), the uncalled CNVs were visible on Interpret but the protocol of the study precluded them from being called. We have described an improved method to investigate ID/DD samples, providing critical information on not just CNVs, but SNVs and Indels as well.




Results I

Overview of Study

Type of Sample Number of Samples
Control Samples (Positive and Negitive) 72
Research Samples (Donated from Clinical Labs) 142
Total Number of Samples 214


Table 1: The types of samples used in the study to test the reagents and software. These were designed to test all aspects of the system namely, the SNV / Indel, CNV (large and small) and LOH performance. The samples included control and research samples from genetic laboratories, where a pathogenic CNV, SNV, Indel or stretch of LOH had been identified. These were identified by microarrays (CNVs, LOH) or sequencing (SNV, Indel).


Figure 1

Figure 1: Mutation in GJB2 gene on chromosome 13


Figure 2

Figure 2: ANKRD11: c2408_2412del identified on chromosome 16

CNV - Duplication

Figure 3

Figure 3: A 65.8Kb duplication on chromosome 2. Top panel shows the CytoSure NGS assay viewed with Interpret. Bottom panel is a Microarray analysis.

CNV - Duplication and LOH

Figure 4 CNV Duplication and LOHFigure 4: 10.39Mb LOH and a 213.75Kb duplication on chromosome 7. Top panel shows the CytoSure NGS assay viewed with Interpret. Bottom panel is a Microarray analysis.

CNV - Large Deletion and LOH

Figure 5 Large deletion and LOH

Figure 5: A 5.91MB deletion on chromosome 1. Top panel shows the CytoSure NGS assay viewed with Interpret. Bottom panel is a Microarray analysis.

CNV - Small Deletion

Figure 6 Small Deletion

Figure 6: A 150b intragenic deletion on chromosome X. Top panel shows the CytoSure NGS assay viewed with Interpret. Bottom panel is a Microarray analysis.

Aberration Calls Rate
SNV/Indels 43/43 100%
Large CNVs 36/37 97.3%
Small CNVs 49/51 96%
Total CNVs 85/88 97%
LOH (>5Mb) 104/105 99%
Mosaic (>5Mb) 5/5 100%


Table 2: Illustrating the results obtained in the study, with the number of samples where the pathogenic aberration was called by the software and the % sensitivity for each type.

Results II

Mosaic Samples

A small number of mosaic samples with CNVs were included. The detection of these is dependent on the size of the aberration and the level of mosaicism. Our criteria for including mosaic calls was that the aberration be >5Mb in size and a mosaicism of 50% or more.
Figure 7

Figure 7: mosaic deletion of 7Mb called in chromosome 15.

Figure 8

Figure 8: Although the 10% Trisomy 9, represented in the aneuploidy plot above, was not called in our protocol, as the mosaicism was below 50% - we were still able to visualise the aberration manually.


This study, using a large range of clinical research samples, demonstrates that CytoSure™ Constitutional NGS solution is able to detect pathogenic CNVs and LOH with a performance on par with microarrays – as demonstrated in the Results I section. These can be large CNVs (Figure 5), megabases in size, or small intragenic CNVs (Figure 6). We also called 100% of reported pathogenic SNVs/Indels, showing the precision and sensitivity of the assay. In the Results II section we discussed the ability of the assay to detect CNVs in low percentage mosaic samples, a result of the bait design and accuracy of Interpret software.

CytoSure Constitutional NGS’ ability to call both SNVs and CNVs gives this assay a larger scope in calling genetic aberrations than in microarrays alone.




  • Centre hospitalier universitaire de Sherbrooke, Quebec, Canada
  • All Wales Medical Genetics Service, Cardiff, UK
  • EGL Genetics, Georgia, USA
  • Clinical Genome Resource (ClinGen)
  • Prof. Matthew Hurles, FMedSci, FRS, Wellcome Sanger Institute


CytoSure: For Research Use Only; Not for Diagnostic Procedures. © Oxford Gene Technology IP Limited – 2020

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