Oxford Nanopore sequencing to handle missing heritability in rare diseases | LC26

Abstract
Despite the widespread use of short-read whole-genome sequencing (SR-WGS), more than half of patients with suspected rare diseases remain without a molecular diagnosis, even after years of extensive investigations. To address this diagnostic gap, we integrated Oxford Nanopore Technologies (ONT) long-read whole-genome sequencing (LR-WGS) into the research workflow of the Bambino Gesù Children’s Hospital Undiagnosed Patient Program, resulting in a substantial enhancement of our discovery capabilities. The adoption of ONT enabled the resolution of previously unsolved cases by providing access to genomic features that are largely inaccessible to SR-WGS. In particular, ONT sequencing allowed comprehensive investigation of challenging ‘dark’ genomic regions, accurate allelic phasing, and native DNA methylation profiling from a single assay. These combined capabilities proved critical for uncovering pathogenic mechanisms underlying complex phenotypes and elusive Mendelian disorders. In this report, we present a series of representative use cases in which ONT LR-WGS was pivotal in identifying disease-causing molecular events in patients who had remained undiagnosed after conventional genomic testing. In addition to variant discovery, we explored the utility of long-read-derived DNA methylation profiling as a functional assay to support variant interpretation and clinical classification. Our preliminary data clearly demonstrates its advantages over SR-WGS in detecting rare and functionally relevant variants, including structural variations and tandem repeats, and concurrent DNA methylation profiling. These findings highlight the unique strengths of ONT long-read sequencing as a versatile tool for clinical genomics research, supporting its integration into our multiomic diagnostic workflow.

Biography
Cecilia Mancini is an experienced researcher with a background in molecular biology and more than 10 years of experience in medical genetics and functional genomics. She received her academic training at the University of Turin, where she earned a PhD in Human Genetics, developing expertise in the molecular mechanisms underlying neurogenetic disorders. Her current research focuses on genomic and gene expression analysis in rare diseases. She has extensive experience with next-generation sequencing and high-throughput approaches, enabling comprehensive characterization of genetic variation.