Integrating copy number, methylation, and fusion detection in one assay: A Nanopore sequencing approach for pediatric and adults' rare tumors diagnosis
Recent advances in sequencing technologies have significantly refined tumor characterization by enabling detailed analysis of genetic alterations, transcriptomic profiles, and DNA methylation patterns. Combined with pathological data, these molecular insights have become essential for accurate tumor classification at diagnosis, allowing for more precise prognostic assessment and the implementation of the most appropriate therapeutic strategies. Currently, obtaining this comprehensive molecular information requires the use of multiple techniques, often leading to extended turnaround times and high cost. A key challenge is therefore the development of an integrated method capable of simultaneously detecting copy number variations, structural rearrangements, and methylation-based classifications in a time-efficient manner. In this study, we applied Nanopore long-read sequencing combined with adaptive sampling to a series of 20 pediatric and rare tumors previously characterized using conventional short-read panels and RNA sequencing. Our approach successfully detected all clinically relevant and classifying structural variants, including gene fusions and internal tandem duplications, previously identified by traditional short read sequencing. In addition, we generated high-quality genome-wide copy number profiles and methylation data from the same DNA sample, enabling integrated molecular classification. These findings demonstrate the strong concordance of this unified method with standard approaches and highlight the value of Nanopore sequencing as a powerful and time-efficient tool for comprehensive tumor profiling. Requiring only a single sample and offering rapid technical processing, this approach aligns with the clinical objective of accelerating diagnostic workflows and improving patient care. Overall design: Nanopore adaptive sequencing of 20 fresh frozen tumor samples. DNA libraries were prepared using the SKQ-LSK114 kit (Oxford Nanopore Technologies), according to the manufacturer's instructions.