The utility of long-read whole genome sequencing in a pediatric cohort with unresolved rare genetic disorders

Abstract

Introduction: Rare genetic disorders remain a major diagnostic challenge, particularly in pediatric populations. Despite advances in next-generation sequencing, including whole-exome sequencing and short-read whole-genome sequencing, diagnostic yield typically ranges from 25–50%, leaving many patients without a definitive molecular diagnosis. Long-read whole-genome sequencing (lrWGS) has emerged as a complementary approach capable of resolving variant classes that are difficult to detect using short-read technologies. This study evaluated the clinical utility of lrWGS for improving diagnostic resolution in pediatric patients with suspected Mendelian disorders. Methods: A cohort of 107 pediatric patients was analyzed, including 87 previously unresolved cases and 20 positive-control cases with known molecular diagnoses. An in-house long-read bioinformatics pipeline was developed and validated using benchmark datasets. Comparative analyses between Oxford Nanopore lrWGS and short-read sequencing were performed, and selected variants were confirmed using orthogonal validation methods, including Sanger sequencing and digital droplet PCR. Results: Benchmarking demonstrated strong performance for single-nucleotide variants and structural variants, while indel detection showed comparatively lower accuracy. lrWGS confirmed 18 of 20 variants in the validation cohort (90% concordance). Application to unresolved cases resolved 8 of 87 patients, yielding an added diagnostic rate of 9.2% and increasing the overall diagnostic rate to 24.5% (26/107). Resolved cases were grouped into two categories: (1) variants missed due to technical limitations requiring improved resolution (n=5), including a deletion in KCNQ2, de novo inversions in SMARCC2 and CSMD1, a repeat expansion in FXN, and a duplication in SCNN1B; and (2) variants with challenging clinical interpretation resolved through reanalysis (n=3), including a single-nucleotide variants in EFTUD2, SMAD2, and ADA. Conclusion: lrWGS provides meaningful incremental diagnostic value in previously unresolved pediatric rare disease cases and is particularly effective for detecting complex variant classes. These findings support the integration of long-read sequencing as a complementary second-tier diagnostic approach in precision medicine workflows. Future integration of methylation analysis may further enhance its diagnostic utility.

Date of Award	2026
Original language	American English
Awarding Institution	HBKU College of Health & Life Sciences