Towards routine long-read sequencing for rare disease: a national pilot study on chromosomal rearrangements

BackgroundClinical genetic laboratories often require comprehensive analysis of chromosomal rearrangements/structural variants (SVs) which can range from gross chromosomal events, such as translocations and inversions, to supernumerary ring/marker chromosomes, and small deletions or duplications. To fully understand the complexity of a specific event and its associated clinical consequences, it is imperative to locate the breakpoint junctions and to resolve the derivative chromosome structure. This task, however, often surpasses the capabilities of conventional short-read sequencing technologies. In contrast, emerging long-read sequencing techniques present a compelling alternative for clinical diagnostics. MethodsHere, the Genomic Medicine Sweden Rare Diseases (GMS-RD) consortium explored the utility of HiFi Revio long-read whole genome sequencing (lrGS) for clinical digital karyotyping of SVs nationwide. The first 16 samples included in this study were collected from all health care regions in Sweden. We established a national pipeline and a shared variant database for variant calling and filtering. The included validation samples cover a spectrum of simple and complex SVs including inversions, translocations and copy number variants. ResultsThe results from the lrGS analysis match the reported karyotype for 14/16 individuals and 12 known SVs were mapped at nucleotide resolution. A complex rearrangement on chromosome 15 was identified only through read depth analysis and two chromosome 21 rearrangements remained undetected, one of which was mosaic. The average read length ranged from 8.3-18.8 kb and the coverage was >20x for all samples. De novo assembly resulted in a limited number of contigs per individual (N50 range 6-86 Mb) clearly separating the two alleles in most cases, enabling direct characterization of the chromosomal rearrangements. ConclusionsIn a national pilot study, we successfully demonstrated the utility of HiFi Revio lrGS as a clinical analysis of chromosomal rearrangements. Based on our results we propose a five-year plan for the wider implementation of lrGS for rare disease diagnostics in Sweden.