Combining long-read DNA and RNA sequencing to enhance molecular understanding of structural variations leading to copy gains.

Structural variants (SVs) significantly contribute to human disease, but their complexity often makes accurate characterization difficult with conventional methods. Advances in long-read sequencing (LRS) offer potential by spanning kilobases and directly resolving SVs. In this study, we examined two individuals with unresolved SVs. LRS on both DNA and cDNA provided single-base resolution of all breakpoint junctions, revealing detailed rearrangement structures and underlying mechanisms. Transcriptomic analyses identified abnormal fusion transcripts and clarified their functional consequences, including haploinsufficiency and potential dominant-negative effects. In one case, a triplication affecting the ZMYM2 gene was precisely mapped, revealing a truncated variant that may escape nonsense-mediated decay. In the second case, a highly complex reciprocal translocation involving RERE and FHAD1 disrupted RERE expression, with Hi-C data showing minimal impact on enhancer-promoter interactions. Due to their complexity, these SVs were not fully resolved by standard methods. By integrating LRS with transcriptomic and chromosomal conformation analyses, we provided a comprehensive understanding of SV formation and its pathogenic impact. Our findings emphasize the need for advanced genomic approaches to resolve complex SVs, enhance diagnostic accuracy, and inform clinical management.