New advancements in long-read sequencing (LRS) technologies are showing promise for providing conclusive diagnoses for patients with rare diseases, where traditional genetic testing often falls short. A recent study reveals the efficacy of a novel filtration strategy using LRS data, which has the potential to transform clinical genetic testing.
Conducted by researchers at various institutions, the study was published on March 14, 2025, and presents findings from their refined approach to detecting pathogenic genomic and epigenomic variations. This method allows for enhanced identification of both small and large genetic variants, as well as abnormal episignature disease profiles, thereby addressing the challenge of undiagnosed cases within the rare disease community.
Rare diseases, which affect approximately 400 million individuals worldwide, often have complex genetic origins. The overwhelming diversity of pathogenic changes — including single nucleotide variants (SNVs), structural variants, and methylation changes — complicates the diagnostic process, which can take up to six years to yield answers through traditional short-read sequencing methods. Although some advancements have been made, over half of the patients with suspected genetic disorders do not receive a clear diagnosis.
This study leverages LRS, particularly Oxford Nanopore technology, to target at least 30X coverage and achieve average N50 read lengths of 12 kilobases. "The results demonstrate how LRS holds great potential for enhancing clinical testing for rare diseases by enabling the discovery of both known and novel disease variations," say the authors of the article.
By optimizing their workflow, researchers have seen significant advancements. They report the identification of various pathogenic variants across genomic and epigenomic landscapes, with some results including the detection of all known pathogenic variants within specific, well-characterized patient cohorts. Among 76 positive control samples, they successfully identified all pathogenic single nucleotide, structural, and methylation variants, which cements the viability of the proposed methodologies.
Further, the filtration approach applied to 51 patients previously deemed undiagnosed led to substantial discoveries; 10% of these patients received new diagnoses based on their methylation profiles. One significant finding under the study was at the spinal muscular atrophy (SMA) locus, which is recognized for causing severe neuromuscular challenges.
Analysis revealed varying degrees of methylation modification specific to SMA patients (0-15% modification), carriers (50-70%), and non-carriers (98-100%) within certain exon-intron boundaries of the SMN1 gene. “We propose the methylation tag could serve as both a diagnostic indication and carrier status identification marker for SMA,” the authors stated.
The study also highlighted the importance of their funnel-down filtration strategy, which reduced the number of candidates for manual inspection by 58.8% for copy number variations and 99.2% for structural variations, significantly streamlining the diagnostic process.
Among those 51 undiagnosed patients tested, 41% had already undergone multiple genetic tests, with the majority having undergone chromosomal microarray testing as part of their diagnostic odyssey. These figures underline the complexity of rare disease diagnosis and the need for improved technologies like LRS.
Unique findings for individual patients included pathogenic deletions identified at locations 2q11.1-q11.2 and 16q23.3. Another patient was found to have Hunter McAlpine syndrome due to duplication at 5q35.2-q35.3 involving the NSD1 gene. The combination of genomic deletions and methylation profiling illustrated how LRS could potentially provide comprehensive insights where previous methods failed.
Despite presenting promising results, the authors caution about the challenges the field still faces, such as filtering out non-coding variants and the need for functional validation of certain identified variants. They emphasized though, the potential of LRS to serve as a unified approach for genetic testing, effectively integrating diagnostic techniques for rare diseases.
By establishing this new framework and utilizing advanced genomic technologies, researchers are setting the stage for improved outcomes for patients grappling with rare diseases globally. The study encourages the adoption of comprehensive genetic testing methods within clinical settings to minimize diagnostic delays and provide effective management strategies.
Through continued innovations, long-read sequencing could play a transformative role in the future of genetic research and disease management, paving the way for earlier interventions and improved patient care.