Resolving a complex structural variant case study
Recent research has demonstrated that complex structural variations (cxSVs) — which involve three or more breakpoint junctions — are considerably more abundant and diverse than previously appreciated, with an average of 14 cxSVs detected in developmental disorder samples1. The advent of high-throughput genomic technologies has made the identification of cxSVs possible; however, detailed breakpoint analysis is still challenging, even for modern sequencing and microarray platforms.
At the University of Cambridge, UK, Alba Sanchis-Juan and colleagues are studying the association of cxSVs with Mendelian disease2. Using short-read sequencing technology they screened 1,342 samples from individuals with neurodevelopmental or retinal disorders to identify cxSVs that overlapped with clinically-important, disease-associated genes. Of the four samples identified, the nature of the variant could be resolved in all but one sample using a combination of short-read sequencing, Sanger sequencing and microarray analysis. Data for the unresolved sample, which came from a female neonate who presented with foetal bradycardia (low heart rate), suggested two alternative models of cxSV structure, in which either one or two disrupted copies of the gene CDKL5, associated with early infantile epileptic encephalopathy, were present (Figure 1).
To characterise the true nature and likely pathogenicity of the cxSV, the team turned to the long read sequencing capabilities offered by nanopore technology.
‘Long-read whole genome sequencing is the best technology to look at breakpoint analysis in repetitive regions’3.
Using a low-pass whole genome sequencing approach on the MinION, a minimum of 4-fold coverage was achieved for all breakpoints, with an average read length of 8kb. The long reads allowed phasing of the aberrant locus to be determined, identifying paternal inheritance. The phased reads also established the presence of an intact and disrupted copy of the CDKL5 gene, providing evidence to support the proposed model 1 (Figure 1). Furthermore, nanopore sequencing confirmed all the novel breakpoint junctions that could not be elucidated using Sanger sequencing due to the repetitive nature of the DNA, which precluded the design of specific primers. Subsequent gene expression analysis demonstrated that both CDKL5 alleles are expressed in the sample, indicating that the variant is likely benign.
This case study was taken from the clinical white paper.
1. Collins, R.L., et al. Defining the diverse spectrum of inversions, complex structural variation, and chromothripsis in the morbid human genome. Genome Biol. 18, 36 (2017).
2. Sanchis-Juan, A. et al. Complex structural variants in Mendelian disorders: identification and breakpoint resolution using short- and long-read genome sequencing. Genome Med. 10(1):95 (2018).
3. Sanchis-Juan, A. Complex structural variants resolved by short-read and long-read whole genome sequencing in Mendelian disorders. Presentation. Available at: https://nanoporetech. com/resource-centre/complex-structural-variantsresolved-short-read-and-long-read-wholegenome-0 [Accessed: 30 October 2018]