Genetic testing has advanced enormously in the past decade, mainly thanks to short-read sequencing (SRS) technologies – such as Illumina platforms – which read DNA in very small fragments. These systems work well for identifying many disease-causing mutations and are now standard in clinical diagnostics. However, they still struggle with several important tasks: detecting large structural changes in DNA, finding variants hidden deep inside introns, resolving genes with repetitive regions or pseudogenes, and achieving complete coverage in difficult genomic areas. In diagnostic practice, this means some pathogenic variants are still being missed.
Long-read sequencing (LRS) technologies, such as those developed by Oxford Nanopore Technologies (ONT), promise to overcome many of these limitations, as they can read much longer DNA fragments – even thousands of bases at once – allowing complex genetic regions to be explored more accurately and structural variations to be seen directly. This study aimed to determine whether the ONT LRS technology platform is reliable enough to be used in clinical diagnostics, particularly for genetically heterogeneous disorders.
The primary objective of the study was to validate ONT LRS for clinical use in alignment with the EU Health Technology Assessment (HTA) regulation. To do this, the researchers tested 509 DNA samples that had already undergone routine clinical genetic analysis. Of note, the study was conducted using the CE-IVD panels HEVA Pro, CARDIO Pro, BRaCA and ClinEX Pro from 4bases, which are compatible with the ONT technology. The results were striking.
The ONT LRS successfully detected 100% of the known pathogenic or likely pathogenic (P/LP) variants that were previously identified by SRS and confirmed via Sanger sequencing, MLPA or quantitative PCR, achieving a diagnostic performance at least equal to the SRS technique currently adopted in clinical practice. Beyond excellent sensitivity, the ONT LRS platform also provided much better coverage in genomic regions that are notoriously hard for SRS technologies and excelled at detecting structural variants (SVs). The study highlights cases where it identified copy-number variants and large deletions that had been completely missed by SRS. Finally, the researchers implemented a “fast-track” workflow capable of delivering results within 24 hours, an attractive option for urgent clinical situations where rapid decisions are needed.
Overall, the study concludes that ONT long-read sequencing, coupled with the 4bases compatible, CE-IVD panels, can match – and in many respects exceed – the diagnostic performance of current short-read technologies, including the currently recognized gold standard that is represented by the Illumina SRS platform. Its ability to unify multiple diagnostic tasks (point mutations, structural variants, CNVs) into a single workflow could reduce complexity and cost while increasing diagnostic yield. This is a bright perspective for the widespread use of LRS and for the market development of the 4bases panels HEVA Pro, CARDIO Pro, BRaCA and ClinEX Pro.
Eager to learn more details? Read the full study!
Mario Urtis, Chiara Paganini, Viviana Vilardo, Antonio Tescari, Samantha Minetto, Claudia Cavaliere, Andrea Pilotto, Carmela Giorgianni, Alessia Cattaneo, Marilena Tagliani, Maurizia Grasso, Alexandra Smirnova, Payam Ebadi, Valentina Barzon, Valentina Favalli, Andrea Bimbocci, Marta Baragli, Alberto Magi, Alessandra Renieri and Eloisa Arbustini
