Background

Phenomena in which specific sequences in the human genome are repeated dozens to thousands of times are known to cause various diseases. Huntington's disease and Spinocerebellar Ataxia (SCA) are representative examples of repeat-expansion disorders (REDs). Until now, Southern blot and Polymerase Chain Reaction (PCR) have been primarily used to diagnose these genetic disorders. However, these methods have been criticized for their near-impossibility in accurately measuring the length of sequences when the repeat regions exceed a certain threshold. Short-read sequencing (SRS), which analyzes the genome in fragments of 100–150 base pairs, has also shown significant technical limitations in reconstructing the exact position and total length of repeat sequences. Without an accurate genome map, predicting individual symptoms and developing personalized treatment strategies have often been delayed.

Key Findings

The LRS-RED consortium has published a summary of the latest methodological and bioinformatics advances for the clinical application of long-read sequencing (LRS) technology. The core technology presented in this report is a sequencing method that does not require PCR amplification. First, the disease-causing regions are precisely excised using the gene scissors CRISPR/Cas9. Then, the entire DNA sequences are read in tens of thousands of units using Single Molecule, Real-Time (SMRT) or Nanopore technology. By omitting the PCR amplification process, the sequencing is performed without damaging the methylation status originally present in the DNA sequence, enabling real-time detection of epigenetic changes critical for gene expression regulation, the researchers added. LRS can fully decode even the fine structural interruptions within repeat sequences, which were previously indistinguishable using existing technologies. Specialized algorithms developed to precisely classify the size and type of complex repeat sequences contribute to improved analytical accuracy. Moreover, LRS can distinguish somatic mosaicism, a phenomenon in which the length of repeat sequences varies among cells within the same patient, at a resolution level. While traditional PCR tests only showed average values by mixing genetic material from multiple cells, LRS effectively visualizes the distribution of mutations at the single-molecule level.

Significance and Outlook

This study offers new hope for patients with undiagnosed rare diseases. Many patients with neurodegenerative diseases who could not be diagnosed with existing tests now have the possibility of receiving a confirmed diagnosis through the introduction of LRS. By accurately identifying the motif of the repeat sequences causing the disease, it is expected to contribute to the development of patient-specific treatment strategies in the future. However, there are practical barriers to entry, such as the high cost of equipment and the requirement for high-quality DNA extraction. Standardization of device performance and the development of advanced software capable of processing large-scale genomic data remain critical tasks. In response, the academic community is developing clinical guidelines that optimally combine LRS with existing diagnostic methods to enhance efficiency.