Background Classification of central nervous system (CNS) tumors has become increasingly complex over the past decade, raising concerns about the availability, feasibility and sustainability of comprehensive molecular diagnostics. We have evaluated nanopore whole genome sequencing (nWGS) as a single workflow to replace multiple diagnostic assays.

Methods We performed nWGS on DNA extracted from 90 adult CNS tumor samples (58 retrospective, 32 prospective) and compared the results to findings from standard of care (SoC) diagnostic work-up. Analysis was done through an automated workflow that consolidated diagnostically and therapeutically relevant genomic alterations, including copy-number variation, structural, and single-nucleotide variants, chromosomal aberrations, gene fusions and methylation-based classification.

Results Nanopore WGS enabled final diagnostic classification in all samples with >15% tumor cell content, requiring ∼3 hours of hands-on library preparation, parallel sample processing, and sequencing times within 72 hours. Methylation-based classification was available within 1 hour and was concordant with the integrated final diagnosis in 89% of cases (80/90). All diagnostically relevant copy-number variations, single-nucleotide variants, and gene fusions were concordant with standard-of-care testing, and MGMT promoter methylation status matched in 94% of cases. In addition, nWGS identified prognostic and potentially actionable variants that were not reported or covered by SoC.

Conclusions Nanopore WGS delivers comprehensive genetic and epigenetic results with a fast turn-around compared to standard methods. This enables efficient, accurate, and scalable molecular diagnostics of CNS tumors using a single platform. Its broad applicability supports its implementation in routine clinical practice and may be extended to other cancer types requiring complex genomic profiling.

F.S. is a co-founder and shareholder of Heidelberg Epignostix GmbH. A.P. became a full-time employee of Heidelberg Epignostix GmbH in December 2024. A.P. and F.S. are inventors on a patent application related to a nanopore sequencing-based method for cancer characterization, filed by Deutsches Krebsforschungszentrum (DKFZ), Universitat Heidelberg, and Oxford Nanopore Technologies PLC (patent application number: 18682016). S.H., H.L. and E.O.V.M. have received reimbursement for travel, accommodation and conference fees to speak at events organized by ONT. The other authors declare no competing interests.

https://www.biorxiv.org/content/10.64898/2026.03.25.714119v2

This work was supported by grants from Norwegian South-Eastern regional health authorities [grant numbers 2021039, 2023059 to S.H., H.L. and E.O.V.M].

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The study was approved by the Regional Ethics Committee for South East Norway, approvals number 388359 and 853700).

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