{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,25]],"date-time":"2026-02-25T13:02:43Z","timestamp":1772024563328,"version":"3.50.1"},"reference-count":33,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2025,11,3]],"date-time":"2025-11-03T00:00:00Z","timestamp":1762128000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003196","name":"Italian Ministry of Health","doi-asserted-by":"publisher","award":["PNC-E3-2022-23683266"],"award-info":[{"award-number":["PNC-E3-2022-23683266"]}],"id":[{"id":"10.13039\/501100003196","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Genes"],"abstract":"<jats:p>Background\/Objectives: Short-read-sequencing (SRS) is currently the standard for genetic testing in inherited human diseases. Intrinsic limitations include PCR dependency, restricted read length, and challenges in identifying structural variants (SVs), copy number variations (CNVs), and intronic small variants (SNVs\/indels). Long-read-sequencing (LRS) enables the sequencing of long DNA molecules, detection of deep intronic variants, rapid testing of few samples, and improved resolution of SVs, CNVs, and SNVs\/indels. We therefore aimed to validate Oxford Nanopore Technologies (ONT) LRS for potential clinical application. Methods: We evaluated the ONT\u2019s ability to detect pathogenic\/likely pathogenic (P\/LP) variants previously identified by SRS and confirmed via Sanger sequencing, Multiplex-Ligation-dependent-Probe-Amplification (MLPA), or quantitative-PCR (qPCR). In total, 509 samples were analyzed, including 393 with P\/LP variants and 116 negative controls. We used CE-IVD panels HEVA pro, CARDIO pro, BRaCA panel, and ClinEX pro (4Bases-CH). Sequencing was performed on MinION, GridION, and PromethION-2 platforms. Data were analyzed using the 4eVAR pipeline. Results: ONT successfully identified all P\/LP variants across the panels (sensitivity 100%); identified a previously missed CNV in ENG gene; precisely defined the breakpoints of a del(13q) (unsuspected and diagnosed as BRCA2 del ex2\u201314); improved the coverage profiles in difficult-to-map regions (e.g., ex1 TGFBR1, PSM2CL); expanded the coverage of out-of-target deep intronic regions; and allowed for the set-up of fast-track tests (&lt;24 h) for urgent clinical needs. Conclusions: Our findings demonstrate that ONT LRS provides diagnostic performance comparable to SRS, with significant advantages in resolving complex and previously undetectable variants. Ongoing developments are further increasing read length, expanding detectable targets, and potential clinical applications.<\/jats:p>","DOI":"10.3390\/genes16111325","type":"journal-article","created":{"date-parts":[[2025,11,3]],"date-time":"2025-11-03T13:22:36Z","timestamp":1762176156000},"page":"1325","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Oxford Nanopore Technologies [ONT] Sequencing: Clinical Validation in Genetically Heterogeneous Disorders"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3289-9228","authenticated-orcid":false,"given":"Mario","family":"Urtis","sequence":"first","affiliation":[{"name":"Centre for Inherited Diseases, Department of Research, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chiara","family":"Paganini","sequence":"additional","affiliation":[{"name":"Centre for Inherited Diseases, Department of Research, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Viviana","family":"Vilardo","sequence":"additional","affiliation":[{"name":"Centre for Inherited Diseases, Department of Research, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-3297-8085","authenticated-orcid":false,"given":"Antonio","family":"Tescari","sequence":"additional","affiliation":[{"name":"Centre for Inherited Diseases, Department of Research, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy"},{"name":"Dipartimento di Medicina Sperimentale, Universit\u00e0 del Salento, 73100 Lecce, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Samantha","family":"Minetto","sequence":"additional","affiliation":[{"name":"Centre for Inherited Diseases, Department of Research, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy"},{"name":"Dipartimento di Medicina Sperimentale, Universit\u00e0 del Salento, 73100 Lecce, Italy"},{"name":"Genetica Medica, Azienda Ospedaliero-Universitaria Senese, 53100 Siena, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Claudia","family":"Cavaliere","sequence":"additional","affiliation":[{"name":"Centre for Inherited Diseases, Department of Research, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Andrea","family":"Pilotto","sequence":"additional","affiliation":[{"name":"Centre for Inherited Diseases, Department of Research, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Carmela","family":"Giorgianni","sequence":"additional","affiliation":[{"name":"Centre for Inherited Diseases, Department of Research, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alessia","family":"Cattaneo","sequence":"additional","affiliation":[{"name":"Centre for Inherited Diseases, Department of Research, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marilena","family":"Tagliani","sequence":"additional","affiliation":[{"name":"Centre for Inherited Diseases, Department of Research, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2611-4295","authenticated-orcid":false,"given":"Maurizia","family":"Grasso","sequence":"additional","affiliation":[{"name":"Centre for Inherited Diseases, Department of Research, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8911-8827","authenticated-orcid":false,"given":"Alexandra","family":"Smirnova","sequence":"additional","affiliation":[{"name":"Centre for Inherited Diseases, Department of Research, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Payam","family":"Ebadi","sequence":"additional","affiliation":[{"name":"4bases Italia srl, 27100 Pavia, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Valentina","family":"Barzon","sequence":"additional","affiliation":[{"name":"4bases Italia srl, 27100 Pavia, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Valentina","family":"Favalli","sequence":"additional","affiliation":[{"name":"4bases Italia srl, 27100 Pavia, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Andrea","family":"Bimbocci","sequence":"additional","affiliation":[{"name":"Department of Information Engineering, University of Florence, 50139 Florence, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marta","family":"Baragli","sequence":"additional","affiliation":[{"name":"Department of Information Engineering, University of Florence, 50139 Florence, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7393-4283","authenticated-orcid":false,"given":"Alberto","family":"Magi","sequence":"additional","affiliation":[{"name":"Department of Information Engineering, University of Florence, 50139 Florence, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0846-9220","authenticated-orcid":false,"given":"Alessandra","family":"Renieri","sequence":"additional","affiliation":[{"name":"Genetica Medica, Azienda Ospedaliero-Universitaria Senese, 53100 Siena, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2948-7994","authenticated-orcid":false,"given":"Eloisa","family":"Arbustini","sequence":"additional","affiliation":[{"name":"Centre for Inherited Diseases, Department of Research, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,3]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"719","DOI":"10.1016\/j.ajhg.2019.08.009","article-title":"A Randomized, Controlled Trial of the Analytic and Diagnostic Performance of Singleton and Trio, Rapid Genome and Exome Sequencing in Ill Infants","volume":"105","author":"Kingsmore","year":"2019","journal-title":"Am. J. Hum. Genet."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1319","DOI":"10.1038\/s41431-021-00924-8","article-title":"Solving the unsolved rare diseases in Europe","volume":"29","author":"Graessner","year":"2021","journal-title":"Eur. J. Hum. Genet."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"e2018109","DOI":"10.1001\/jamanetworkopen.2020.18109","article-title":"Genome Sequencing as a Diagnostic Test in Children with Unexplained Medical Complexity","volume":"3","author":"Costain","year":"2020","journal-title":"JAMA Netw. Open"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"344","DOI":"10.1093\/eurheartj\/ehae747","article-title":"Integration of genetic testing into diagnostic pathways for cardiomyopathies: A clinical consensus statement by the ESC Council on Cardiovascular Genomics","volume":"46","author":"Elliott","year":"2025","journal-title":"Eur. Heart J."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"61","DOI":"10.1038\/nmeth.1527","article-title":"Limitations of next-generation genome sequence assembly","volume":"8","author":"Alkan","year":"2011","journal-title":"Nat. Methods"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1347","DOI":"10.1038\/s41587-020-0538-8","article-title":"A robust benchmark for detection of germline large deletions and insertions","volume":"38","author":"Zook","year":"2020","journal-title":"Nat. Biotechnol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1611676","DOI":"10.3389\/pore.2024.1611676","article-title":"Can long-read sequencing tackle the barriers, which the next-generation could not? A review","volume":"30","author":"Valcz","year":"2024","journal-title":"Pathol. Oncol. Res."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Kaplun, L., Krautz-Peterson, G., Neerman, N., Schindler, Y., Dehan, E., Huettner, C.S., Baumgartner, B.K., Stanley, C., and Kaplun, A. (2025). ONT in Clinical Diagnostics of Repeat Expansion Disorders: Detection and Reporting Challenges. Int. J. Mol. Sci., 26.","DOI":"10.3390\/ijms26062725"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"843","DOI":"10.1016\/j.jmoldx.2024.06.001","article-title":"Variant Detection in 3\u2032 Exons of PMS2 Using Exome Sequencing Data","volume":"26","author":"Mistry","year":"2024","journal-title":"J. Mol. Diagn."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1540","DOI":"10.1158\/0008-5472.CAN-20-1959","article-title":"The BRCA1 Pseudogene Negatively Regulates Antitumor Responses through Inhibition of Innate Immune Defense Mechanisms","volume":"81","author":"Han","year":"2021","journal-title":"Cancer Res."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Kringen, M.K., Stormo, C., Grimholt, R.M., Berg, J.P., and Piehler, A.P. (2012). Copy number variations of the ATP-binding cassette transporter ABCC6 gene and its pseudogenes. BMC Res. Notes, 5.","DOI":"10.1186\/1756-0500-5-425"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"42","DOI":"10.1186\/s13073-023-01194-3","article-title":"Applications of long-read sequencing to Mendelian genetics","volume":"15","author":"Mastrorosa","year":"2023","journal-title":"Genome Med."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Amarasinghe, S.L., Su, S., Dong, X., Zappia, L., Ritchie, M.E., and Gouil, Q. (2020). Opportunities and challenges in long-read sequencing data analysis. Genome Biol., 21.","DOI":"10.1186\/s13059-020-1935-5"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"599","DOI":"10.1101\/gr.280041.124","article-title":"Unraveling the hidden complexity of cancer through long-read sequencing","volume":"35","author":"Li","year":"2025","journal-title":"Genome Res."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"428","DOI":"10.1016\/j.ajhg.2025.01.002","article-title":"Advancing long-read nanopore genome assembly and accurate variant calling for rare disease detection","volume":"112","author":"Negi","year":"2025","journal-title":"Am. J. Hum. Genet."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1186\/s40246-023-00522-3","article-title":"The application of long-read sequencing in clinical settings","volume":"17","author":"Oehler","year":"2023","journal-title":"Hum. Genom."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"359","DOI":"10.1038\/s10038-019-0569-5","article-title":"A 12-kb structural variation in progressive myoclonic epilepsy was newly identified by long-read whole-genome sequencing","volume":"64","author":"Mizuguchi","year":"2019","journal-title":"J. Hum. Genet."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1215","DOI":"10.1038\/s41588-019-0459-y","article-title":"Long-read sequencing identifies GGC repeat expansions in NOTCH2NLC associated with neuronal intranuclear inclusion disease","volume":"51","author":"Sone","year":"2019","journal-title":"Nat. Genet."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s13073-024-01419-z","article-title":"Clinical evaluation of long-read sequencing-based episignature detection in developmental disorders","volume":"17","author":"Geysens","year":"2025","journal-title":"Genome Med."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1038\/gim.2017.86","article-title":"Long-read genome sequencing identifies causal structural variation in a Mendelian disease","volume":"20","author":"Merker","year":"2018","journal-title":"Genet. Med."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"2500","DOI":"10.1038\/s41467-025-57695-9","article-title":"Long read sequencing enhances pathogenic and novel variation discovery in patients with rare diseases","volume":"16","author":"Sinha","year":"2025","journal-title":"Nat. Commun."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1825","DOI":"10.1101\/gr.279158.124","article-title":"Long-read DNA and cDNA sequencing identify cancer-predisposing deep intronic variation in tumor-suppressor genes","volume":"34","author":"Gulsuner","year":"2024","journal-title":"Genome Res."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"108365","DOI":"10.1016\/j.isci.2023.108365","article-title":"Uncovering the true features of dystrophin gene rearrangement and improving the molecular diagnosis of Duchenne and Becker muscular dystrophies","volume":"26","author":"Ling","year":"2023","journal-title":"iScience"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"114","DOI":"10.1186\/s13073-023-01270-8","article-title":"Beyond the exome: Utility of long-read whole genome sequencing in exome-negative autosomal recessive diseases","volume":"15","author":"AlAbdi","year":"2023","journal-title":"Genome Med."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"313","DOI":"10.1200\/OP-24-00453","article-title":"Multidisciplinary Management of Pregnancy-Associated Breast Cancer","volume":"21","author":"Roesch","year":"2025","journal-title":"JCO Oncol. Pract."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2541","DOI":"10.1002\/ajmg.a.62349","article-title":"An approach to rapid characterization of DMD copy number variants for prenatal risk assessment","volume":"185","author":"Chin","year":"2021","journal-title":"Am. J. Med. Genet. A"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Mendes de Almeida, R., Tavares, J., Martins, S., Carvalho, T., Enguita, F.J., Brito, D., Carmo-Fonseca, M., and Lopes, L.R. (2017). Whole gene sequencing identifies deep-intronic variants with potential functional impact in patients with hypertrophic cardiomyopathy. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0182946"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1186\/s40246-024-00697-3","article-title":"Long-read sequencing enables comprehensive molecular genetic diagnosis of Fabry disease","volume":"18","author":"Yao","year":"2024","journal-title":"Hum. Genom."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1007\/s10689-021-00236-2","article-title":"Use of sanger and next-generation sequencing to screen for mosaic and intronic APC variants in unexplained colorectal polyposis patients","volume":"21","author":"Elsayed","year":"2022","journal-title":"Fam. Cancer"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1045","DOI":"10.1002\/humu.22082","article-title":"Deep intronic APC mutations explain a substantial proportion of patients with familial or early-onset adenomatous polyposis","volume":"33","author":"Spier","year":"2012","journal-title":"Hum. Mutat."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"914","DOI":"10.1101\/gr.279323.124","article-title":"Integration of transcriptomics and long-read genomics prioritizes structural variants in rare disease","volume":"35","author":"Jensen","year":"2025","journal-title":"Genome Res."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"779","DOI":"10.1016\/j.ajhg.2025.02.006","article-title":"Clinical validation of RNA sequencing for Mendelian disorder diagnostics","volume":"112","author":"Zhao","year":"2025","journal-title":"Am. J. Hum. Genet."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"469","DOI":"10.1038\/s41588-024-02067-0","article-title":"Synchronized long-read genome, methylome, epigenome and transcriptome profiling resolve a Mendelian condition","volume":"57","author":"Vollger","year":"2025","journal-title":"Nat. Genet."}],"container-title":["Genes"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4425\/16\/11\/1325\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,11,3]],"date-time":"2025-11-03T13:37:36Z","timestamp":1762177056000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4425\/16\/11\/1325"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,11,3]]},"references-count":33,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2025,11]]}},"alternative-id":["genes16111325"],"URL":"https:\/\/doi.org\/10.3390\/genes16111325","relation":{},"ISSN":["2073-4425"],"issn-type":[{"value":"2073-4425","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,11,3]]}}}