Abstract
BACKGROUND: Determining the correct structure of large, interspersed duplications and related complex genomic rearrangements in genetic disease is critical when establishing causal roles and requires a technology able to span the entire duplicated segment(s) on single molecules. We assessed the use of Bionano optical genome mapping (OGM) for this purpose. METHODS: We combined OGM, Illumina short-read sequencing and fluorescence in situ hybridisation (FISH) to characterise three large interspersed duplications/triplications, and used the deepC algorithm to predict impact on local topologically-associating domains (TADs), assisting functional interpretation. RESULTS: Case 1 harboured paired interspersed duplications (244/323 kb) on chromosome 13. By analysing multiple molecules > 300 kb completely spanning the smaller duplication, we unambiguously determined the correct structure, which potentially alters the TAD containing FGF9, a candidate gene. In Case 2, involving a child with hypertrichosis and gingival hyperplasia (HTC), duplications on chromosomes 16 (2.01 Mb) and 17 (564 kb) were linked on short-read sequencing. By obtaining three OGM molecules spanning the 564 kb segment, we deduced that a t(16;17) translocation was present, which we confirmed by FISH. This interpretation has important implications for clinical risk and highlights KCNJ2 as a potential driver of the HTC3 locus at 17q24.3. Case 3 involved a complex chromoanasynthesis event on chromosome 20. OGM readily resolved all but two of 12 alternative structures; however full resolution required reads to span a 627 kb duplication, which we could not achieve consistently. CONCLUSIONS: OGM represents a powerful tool for disambiguating complex structural variants, but requires multiple individual reads to completely span the duplicated segment. In our hands the upper size of duplications that could be resolved was ~ 550 kb. Deducing the correct configuration is critical both for mechanistic understanding of pathogenesis and accurate recurrence risk counselling. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13073-025-01571-0.