Utility of long-read whole-genome sequencing across diverse clinical genomic investigations: a single- center experience | LC26

Abstract
Long-read sequencing (LRS) is increasingly adopted in clinical genomics, driven by advances in sequencing chemistry and analytical pipelines that have substantially reduced historical error rates. Long-read whole-genome sequencing (LR-WGS) enables comprehensive detection of diverse variant classes, including single nucleotide variants (SNVs), copy number variants (CNVs), structural variants, repeat expansions, and epigenetic modifications such as DNA methylation. Here, we present a single-center experience applying LR-WGS across a range of clinical indications. Between 2023 and 2026, a total of 126 genomes were analyzed. Whole-genome libraries were prepared using the standard Oxford Nanopore Technologies (ONT) ligation protocol and sequenced on the PromethION 24 platform, generating 90–100 Gb per sample (~30× coverage). POD5 files were basecalled using Guppy in high-accuracy (HAC, 400 bps) mode, and reads were aligned to the hg38 reference genome using minimap2 within the Sentieon framework. Short tandem repeat (STR) analysis was performed with Straglr, and variant call format (VCF) files were annotated using Geneyx Analysis. Clinical applications included haplotype analysis (n=20), preimplantation genetic diagnosis (PGD)-related haplotyping (n=13), imprinting disorder validation (n=4), modifier variant analysis (n=15), de novo phasing (n=2), undiagnosed cases (38 singletons and 2 trios), pseudogene-region variant validation (n=4), and repeat expansion validation (n=24). Repeat expansion detection demonstrated 94% sensitivity with accurate allele sizing, while all imprinting disorder cases exhibited distinct methylation signatures. Among previously undiagnosed cases, LR-WGS achieved a diagnostic yield of 15%, identifying pathogenic variants including complex structural rearrangements, deep intronic variants, regulatory region deletions, and variants in challenging genomic regions. In conclusion, LR-WGS is a powerful and versatile approach for resolving complex genomic variation and improving diagnostic yield, with particular strength in repeat expansion detection, methylation profiling, variant phasing, and analysis of difficult genomic regions such as pseudogenes. These findings support the integration of LR-WGS as a complementary modality in routine clinical diagnostics.

Biography
Hagar Mor-Shaked is the head of the next-generation sequencing (NGS) analysis team at Hadassah, Principal Investigator at the Faculty of Medicine, Hebrew University, and Chief Product Officer of Geneyx. Hagar is one of the leading and most experienced clinical genetic experts in Israel. She is an esteemed clinical genetic analyst, with over 10 years of experience in molecular genetics, genomics, and bioinformatics in a clinical and academic setting. As head of bioinformatics at the genetic department of Hadassah-Hebrew University Medical Center, she gained expertise in using different NGS methodologies for diagnosing rare diseases, analyzed thousands of clinical exome and genome sequencing tests, and established the largest genomic database in Israel. In addition, Hagar has published dozens of high-quality academic papers. Hagar holds an MSc in genetic counseling and a PhD in human genetics from the Hebrew University of Jerusalem.