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

Conventional molecular diagnostics for imprinting disorders rely on sequential DNA-intensive assays, which are expensive, time-consuming, and often insufficient for detecting mosaicism, resulting in suboptimal clinical management. Here, we present a targeted long-read sequencing strategy that enables the integrated detection of DNA methylation, copy-number variants, and sequence variants in a single assay for Beckwith-Wiedemann spectrum (BWSp), a genomic imprinting disorder caused by genetic or epigenetic alterations affecting imprinting control regions 1 and 2 (IC1/IC2) within the 11p15.5 locus. We evaluated three Oxford Nanopore Technologies (ONT) workflows: adaptive sampling on MinION (AS-MinION), adaptive sampling on PromethION P2 (AS-P2), and whole-genome sequencing on P2 (WGS-P2). Three cases of BWSp patients were analyzed, including two with mosaic paternal uniparental disomy (pUPD) and one with IC2 loss of methylation (LoM). We compared sequencing output, genome-wide and region-of-interest coverage, IC1/IC2 methylation profiles, and variant concordance with Illumina short read sequencing using the Simplex basecalling model. AS-P2 achieved the highest coverage of target regions while maintaining broad and uniform genome-wide coverage, outperforming AS-MinION and WGS-P2. This dual performance enables efficient and scalable simultaneous genetic and epigenetic analysis in a single sequencing run. Using Simplex basecalling, AS-P2 accurately identified all underlying molecular defects in conventionally characterized samples. In conclusion, AS-P2 enablesa cost-effective and sensitive approach for the molecular diagnosis of imprinting disorders, particularly in cases with mosaic or complex genetic and epigenetic architectures.