[Resolving Genomic Mysteries with Long-read Sequencing]

Short-read next-generation sequencing (srNGS, 50-300 bp) has become the predominant first-line test for diagnosing patients with rare genetic conditions. However, this technology has inherent limitations, including reduced diagnostic yield for structural variants (SVs) and variants located in repetitive or highly homologous genomic regions. Connecting variants over large genomic distances is also challenging using srNGS. In recent years, long-read sequencing (LRS, thousands up to 2 million bp) has emerged as a promising alternative for genomic testing. By enabling the sequencing of longer DNA fragments, LRS improves the analysis of repetitive regions, enhances SV detection, and facilitates the phasing of distant variants. As a result, LRS is becoming an essential tool in genetic laboratories, aiding in the diagnosis of rare genetic disorders, the identification of pathogenic variants undetectable by short-read sequencing, and the improved clinical interpretation of genomic data. The implementation of LRS in genetic diagnostics represents a significant advance, improving both precision and turnaround times. In this review, we examine the principles of LRS in human genetics, its advantages over conventional sequencing methods, and its impact on the diagnosis of genetic disorders, highlighting recent applications in clinical practice.