Long-read sequencing yields 4.5% more diagnoses in unresolved rare disease cases

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Frontiers in Medicine Published June 7, 2026 Medically reviewed June 7, 2026 DOI ↗ By Dr. Julia Lee, PhD · Oncology, Genomics & Drug Development

Key Takeaway

Consider lrWGS for select unresolved rare disease cases, but recognize the modest 4.5% diagnostic yield and limited evidence.

This systematic review evaluated the diagnostic yield of long-read whole genome sequencing (lrWGS) in patients with rare genetic disorders who had inconclusive or negative results from standard testing, including short-read sequencing. The review included 9 studies with a total of 646 previously unresolved cases.

Across these studies, lrWGS provided a definitive diagnosis in 29 individuals, corresponding to a diagnostic yield of 4.5%. Among the identified variants, structural variants (SVs) accounted for the majority (41.67%), followed by combined SV/single-nucleotide variants (20.83%) and methylation changes (16.67%).

The authors assessed risk of bias using the QUADAS-2 tool, but other limitations include the small sample size and potential lack of generalizability beyond the included studies. The review does not report on clinical outcomes beyond diagnostic yield, so the impact on patient management remains unclear.

Despite these limitations, lrWGS shows potential for improving diagnostic rates in previously unresolved rare disease cases, particularly when applied after whole-exome sequencing and combined with advanced tools such as phasing and methylation profiling. However, further research is needed to confirm these findings and assess clinical utility.

Study Details

Study typeMeta analysis

EvidenceLevel 1

PublishedJun 2026

View Original Abstract ↓

BackgroundNearly half of patients with rare genetic disorders remain undiagnosed, which may in part be due to limitations of current short-read sequencing (SRS) approaches in detecting complex genomic alterations. Long-read whole genome sequencing (lrWGS) technologies can address these limitations through enhanced detection of structural variants (SVs), repetitive regions, and epigenetic changes.MethodsTo evaluate the diagnostic yield of lrWGS in patients with rare genetic diseases receiving inconclusive or negative results from standard testing, we searched the PubMed, Science Direct, Scopus, and ProQuest databases to July 2025 for studies applying lrWGS to unresolved rare disease cases and reporting diagnostic outcomes. Risk of bias was assessed using the QUADAS-2 tool.ResultsNine studies involving 646 previously unresolved cases that underwent lrWGS met the inclusion criteria. Of these, 29 individuals (24 unique diagnoses involving 25 genes) received a definitive diagnosis through lrWGS, a diagnostic yield of 4.5%. SVs accounted for the majority of identified variants (41.67%), followed by combined SV/single-nucleotide variants (20.83%), methylation changes (16.67%), and other variant types (copy number variations, indels, and tandem repeats). Most detected variants were in regions typically inaccessible to short-read whole-exome sequencing (WES). lrWGS also enabled phasing and methylation analysis in a single assay, which was valuable for compound-heterozygosity detection and diagnostic interpretation.ConclusionlrWGS shows clear potential for improving diagnostic rates in previously unresolved rare disease cases, particularly when applied after WES and combined with advanced tools such as phasing and methylation profiling. As technologies evolve and become more accessible, lrWGS may increasingly become a first-tier diagnostic approach, especially in phenotypically complex conditions.Systematic Review Registrationhttps://osf.io/y5azb/overview, identifier 10.17605/OSF.IO/Y5AZB.