(A) High-throughput short-read sequencing (SRS) involves the extraction and fragmentation of genomic DNA, adapter ligation, amplification, and massively parallel sequencing, generating millions of short fragments that are aligned to a reference genome. Sequencing can be performed in single-end mode — where each DNA fragment is read from one end only — or paired-end mode, where both fragment ends are sequenced, providing orientation and insert size information that aids in detecting small structural rearrangements. Coverage depth (the number of times a given base is sequenced) and coverage breadth (the proportion of the genome covered) influence variant detection sensitivity. Variant calling algorithms for structural variants utilize distinct signatures: read-pair signals detect discordant spacing or orientation between paired reads, split-read signals identify reads that align partially to two genomic regions, and relies on local increases or decreases in coverage. (B) Long-read sequencing (LRS) platforms, such as Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), generate reads tens to hundreds of kilobases in length, enabling more contiguous genome assembly. PacBio sequencing relies on single-molecule, real-time detection of fluorescently labeled nucleotide incorporation, while ONT senses ionic current changes as DNA or RNA molecules traverse a nanopore. ZMW, zero-mode waveguide. (C) Optical genome mapping provides a high-resolution, sequence-independent view of chromosomal architecture. Ultra–high molecular weight DNA is fluorescently labeled at specific sequence motifs, linearized, and imaged in nanochannels. The resulting pattern of fluorescent labels creates a physical map of the genome. (D) Comparative overview of sequencing technologies: LRS does not require assembly, thus enabling comprehensive DNA structural variant discovery and full-length RNA sequencing to reconstruct complete transcript isoforms. LRS also allows direct epigenetic profiling, as native DNA molecules are sequenced without chemical conversion or amplification, allowing direct detection of DNA methylation and other base modifications from characteristic signal shifts. dNTP, deoxyribonucleoside triphosphate.