Novel Strategy for Structural Variant Genotyping by Short‐Read Genomic Sequencing From Restriction‐Circles: Experimental and Bioinformatics Proof‐of‐Concept

Structural variants (SVs) comprise copy‐number variants (e.g., deletions and duplications) and copy‐neutral SV (e.g., perfect inversions). Short‐read high‐throughput DNA‐sequencing is the established technology for small‐variant calling in clinical genomics, whereas Oxford nanopore or PacBio long‐read NGS technologies improve SV identification but remain challenging to implement in clinical settings. We developed and validated a novel experimental protocol and bioinformatics pipeline for SV genotyping from short‐read whole‐genome sequencing (WGS) of restriction‐circles of DNA. The experimental protocol includes Bcl I‐restriction digestion of genomic DNA, fragment‐ends ligation and short‐read WGS. The bioinformatics pipeline takes advantage of linking pair‐end reads (LPERs), a key operator associating terminal sequences from each restriction fragment. LPERs contain long‐distance haplotype information, essential for SV‐genotyping. Each SV associates with a specific LPER pattern. The approach was applied to a patient with Duchenne muscular dystrophy (DMD) generating proper LPER vertical (8.6x) and horizontal (83%) coverages with good quality standards. SV‐specific LPER quality filters were designed to improve the consistency of SV detection. The approach was validated by verifying a previously characterised DMD ‐genotype in the proband ( DMD _exons_45‐54 deletion and DMD _exons_38‐43 duplication) with our output. Conventional SV genotyping using other SV detection algorithms can still be successfully applied to complete an accurate definition of the rearrangement. Our SV‐genotyping algorithm detected two novel SVs in Xq28, which were verified by direct molecular investigation, and resulted polymorphic in the general population: a 1.4 kb deletion, allelic frequency AF (CI95%) of 0.83 (0.71–0.91) and a perfect inversion with 0.31 (0.08–0.54) originated by recombination of 99% homologous 8.4 kb inverted repeats (IRs). Although IR‐mediated inversions represent a major challenge for WGS‐based genotyping, our approach correctly indicated the absence of haemophilia recurrent inversions. Additionally, our protocol allows application of regular small‐variant calling. A likely benign SNV in F8 ‐intron‐3 was also identified and verified. The potential clinical utility of this approach is supported by its consistency and ability to improve SV genotyping whilst allowing the application of conventional variant calling algorithms.