Accelerated and comprehensive pediatric cancer profiling using whole-genome sequencing with adaptive sampling

Abstract

Abstract Background Timely and comprehensive molecular classification is essential for therapeutic decision-making in pediatric oncology, yet current diagnostic workflows rely on multi-step, time- and resource-intensive testing strategies. We present a standardized whole-genome sequencing approach with adaptive sampling (AS-WGS) using Oxford Nanopore Technologies (ONT) optimized for pediatric cancers, that enables rapid and comprehensive molecular profiling in a single assay. Methods We designed an AS-WGS panel comprising 380 clinically relevant loci that we applied to the sequencing of 36 pediatric cancer and control samples. To support real-time interpretation, we developed an open-source bioinformatic pipeline (nf-core-oncoseq) enabling on-the-fly analysis during sequencing. We compared the AS-WGS results to those obtained by standard clinical assays, including exome and transcriptome sequencing. Results AS-WGS achieved somatic-grade coverage (mean 172X) in target regions while maintaining pan-genomic coverage (18X), thus supporting genome-wide copy number and methylation analyses. AS-WGS reliably detected all classes of clinically actionable alterations including fusion genes (20/20), small variants (SNVs and indels, in 58/59 mutated genes) and large CNVs (20/21). SNVs were robustly captured down to a 5% allelic frequency, similar to clinical exome sequencing. The PCR-free workflow supported methylation-based tumor classification, providing orthogonal confirmation of hematological and solid tumor subtypes in 9/11 acute lymphoblastic leukemias and in 8/12 solid tumors. All clonal alterations were identified with >5 supporting reads from data generated in the first 24 hours of sequencing (20/20 fusions and 47/47 SNVs with allelic frequencies > 25%), with some, such as large CNVs and methylation predictions, detected within the first hour. Conclusions Our results show that applying AS-WGS to pediatric hematological and solid tumors supports the rapid identification of all classes of genomic alterations and enables methylation-based classification in a single assay. Future efforts aimed at clinical translation will establish its position within the current multi-assay diagnostic landscape.