Original articles

Vol. 118: Issue 2 - April 2026

Technical feasibility of a long read, fourth generation sequencing platform in diagnostic profiling of clinical routine samples: a proof-of-concept study

Authors

Domenico Vitale , Lorenzo Colarossi , Gianluca Russo , Claudia Scimone , Domenico Cozzolino , Caterina De Luca , Andrej D. Prjibelski , Alla Mikheenko , Cristina Colarossi , Francesco Delli Muti , Francesco Pepe , Lorenzo Memeo , Giancarlo Troncone , Lucia Anna Muscarella , Umberto Malapelle

Keywords: NGS, tissue samples, bioinformatic pipelines, predictive biomarkers
DOI: 10.32074/1591-951X-1936
Publication Date: 2026-05-13

Summary

Background: Next generation sequencing (NGS) impacted on clinical algorithm of solid tumor patients. A heterogeneous series of NGS platforms have been implemented in clinical practice but challenging handling procedures, high technical costs, and scant affordability on sequencing diagnostic routine specimens can leave behind some patients who could benefit from target drugs. Here, we sought to evaluate technical feasibility of Oxford Nanopore Technologies (ONT) sequencing accurate identification of tumor-associated molecular alterations, in a pilot series of real-world samples.

Methods: We developed a technical workflow adapting the SiRe® NGS panel, originally designed for Ion semiconductor sequencing, on MinION platform (Oxford nanopore technologies), a portable, cost effective long read sequencer. The SiRe® panel enables detection of 568 clinically actionable somatic mutations across six key genes (EGFR, KRAS, NRAS, BRAF, cKIT, PDGFRα) relevant to targeted therapies in several solid tumours. We implemented a multiplexed assay using pooled and barcoded samples, processed on a single MinION flow cell. Performance was benchmarked from a pilot series of nine FFPE samples against Ion Torrent sequencing data. A single liquid biopsy sample was also analyzed testing accuracy of MinION technology.

Results: The adapted ONT workflow demonstrated high concordance rate detecting clinically relevant molecular alterations on short-read fragments, achieving comparable accuracy with standardized second generation NGS platforms on tissue and liquid biopsy samples.

Conclusions: This proof of concept aimed to integrate ONT sequencing into molecular oncology workflows, providing practical, low-cost, and scalable alternative to conventional NGS platforms. The results support the potential of ONT technology to democratize access to precision oncology, particularly in laboratories with limited resources.

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Authors

Domenico Vitale - Department of Public Health, Federico II University of Naples, Via S. Pansini, 5, 80131 Naples, Italy.

Lorenzo Colarossi - Pathology Unit, Department of Experimental Oncology, Mediterranean Institute of Oncology

Gianluca Russo - Department of Public Health, Federico II University of Naples, Via S. Pansini, 5, 80131 Naples, Italy.

Claudia Scimone - Department of Public Health, Federico II University of Naples, Via S. Pansini, 5, 80131 Naples, Italy.

Domenico Cozzolino - Department of Public Health, Federico II University of Naples, Via S. Pansini, 5, 80131 Naples, Italy.

Caterina De Luca - Department of Public Health, Federico II University of Naples, Via S. Pansini, 5, 80131 Naples, Italy.

Andrej D. Prjibelski - Department of Computer Science, University of Helsinki

Alla Mikheenko - UK Dementia Research Institute at UCL, London, UK. Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, London, UK

Cristina Colarossi - Pathology Unit, Department of Experimental Oncology, Mediterranean Institute of Oncology

Francesco Delli Muti - Laboratory of Oncology, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG), Italy

Francesco Pepe - Department of Public Health, Federico II University of Naples, Via S. Pansini, 5, 80131 Naples, Italy

Lorenzo Memeo - Pathology Unit, Department of Experimental Oncology, Mediterranean Institute of Oncology

Giancarlo Troncone - Department of Public Health, Federico II University of Naples, Via S. Pansini, 5, 80131 Naples, Italy

Lucia Anna Muscarella - Laboratory of Oncology, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG), Italy

Umberto Malapelle - Department of Public Health, Federico II University of Naples, Via S. Pansini, 5, 80131 Naples, Italy.

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This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Copyright

Copyright (c) 2026 Società Italiana di Anatomia Patologica e Citopatologia Diagnostica, Divisione Italiana della International Academy of Pathology

How to Cite
Vitale, D., Colarossi, L., Russo, G., Scimone, C., Cozzolino, D., De Luca, C., Prjibelski, A. D., Mikheenko, A., Colarossi, C., Delli Muti, F., Pepe, F., Memeo, L., Troncone, G., Muscarella, L. A., & Malapelle, U. (2026). Technical feasibility of a long read, fourth generation sequencing platform in diagnostic profiling of clinical routine samples: a proof-of-concept study . Pathologica - Journal of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology, 118(2). https://doi.org/10.32074/1591-951X-1936
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