Single-workflow Nanopore whole genome sequencing with adaptive sampling for accelerated and comprehensive pediatric cancer profiling

Timely and comprehensive molecular classification is critical for therapeutic decisions in pediatric oncology. However, current diagnostic workflows rely on multi-step testing and are resource- and time-intensive. We present whole-genome sequencing with adaptive sampling (AS-WGS) protocol using Oxford Nanopore Technologies optimized for pediatric oncology, enabling unified detection of genomic, structural, and epigenomic alterations in a single assay. Applied to 31 pediatric cancer patient samples, AS-WGS achieved high on-target coverage across hundreds of loci of interest for identifying somatic anomalies, while maintaining pan-genomic coverage for copy number assessment and methylome data. We demonstrate that AS-WGS, as a single approach, captures all categories of clinically relevant alterations, including most copy number changes, fusions, and mutations, even subclonal ones. Time stamp analyses revealed that clonal alterations are confidently supported within the first sequencing day, sometimes within the first hours. We developed and reported an open-source bioinformatic pipeline (nf-core-oncoseq) that facilitates streamlined and fully integrated analysis. This approach consolidates complex testing into a single, rapid assay, enabling near real-time cancer characterization. Our findings support AS-WGS as a transformative diagnostic platform for pediatric oncology.