Long-read sequencing as an all-in-one diagnostic approach for genetic testing in cardiovascular diseases

Background Genetic testing represents a fundamental step in the diagnostic workflow for patients affected by genetic cardiovascular diseases. Current testing strategies rely on the integration of multiple technologies to maximize diagnostic yield, with short-read sequencing (SRS) platforms playing a central role. However, the development of long-read sequencing (LRS) technologies holds the promise of providing a comprehensive, all-in-one diagnostic approach capable of overcoming the limitations of existing methodologies. Purpose We assessed the suitability of Oxford Nanopore Technologies (ONT) LRS as a stand-alone diagnostic tool, evaluating its concordance with standard testing for clinically relevant variant detection and its potential to improve diagnostic performance. Methods We analyzed 509 samples using the ONT LRS platform. All samples had previously undergone comprehensive genetic testing workflow comprising Illumina-based SRS, Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), and array comparative genomic hybridization (aCGH). ONT-based results were compared against this reference standard to evaluate diagnostic concordance and added value. Results ONT-based analysis successfully identified all clinically relevant variants detected by the conventional diagnostic workflow. Moreover, it exhibited advantages over SRS in both mapping of difficult genomic regions and detection of copy number and structural variants (Figure). Conclusions Our findings support the implementation of ONT long-read sequencing as a reliable and comprehensive stand-alone technology for genetic testing in cardiovascular diseases, offering enhanced detection of complex and structural genomic variants and the potential to substantially simplify current diagnostic workflows.