Targeted long-read sequencing with adaptive sampling enables the integrated genomic and epigenomic profiling of imprinting disorders

Conventional molecular diagnostics for imprinting disorders rely on sequential DNA-intensive assays, which are expensive, time-consuming, and often fail to detect mosaicism, leading to suboptimal clinical management. Here, we present a targeted long-read sequencing strategy that unifies the detection of methylation, copy-number and sequence variants in a single assay for Beckwith-Wiedemann spectrum (BWSp), a genomic imprinting disorder caused by genetic or epigenetic defects affecting imprinting control regions 1 and 2 (IC1/IC2) within the 11p15.5 cluster. We evaluated three workflows based on Oxford Nanopore Technologies (ONT) platforms: adaptive sampling on MinION (AS-MinION), adaptive sampling on PromethION P2 (AS-P2), and whole-genome sequencing on P2 (WGS-P2). We studied three cases of BWSp, two with mosaic paternal uniparental disomy (pUPD) and one with IC2 loss of methylation (LoM). We compared sequencing output, genome and region-of-interest coverage, IC1/IC2 methylation profiles, and variant concordance with Illumina using Simplex and Herro basecallers. AS-P2 outperformed AS-MinION and WGS-P2 by achieving high coverage of regions of interest while maintaining broad, consistent coverage across the entire genome. This dual capability makes AS-P2 an efficient and scalable solution for genetic and epigenetic analysis in a single run. Compared to conventionally characterized samples, AS-P2 with Simplex basecalling identified all underlying molecular defects accurately. AS-P2 thus provides a cost-efficient and sensitive solution for the diagnosis of imprinting disorders, particularly those with mosaic or complex genetic/epigenetic architectures.