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

Abstract

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 (BPJs) 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 validated HiFi Revio long-read whole genome sequencing (lrGS) for clinical digital karyotyping of SVs nationwide. The first 13 samples included in this study were collected from five of the seven Swedish regions with university hospitals. 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 11/13 individuals and known SVs were mapped at nucleotide resolution. A complex rearrangement on chromosome 15 was identified only through read depth analysis and a mosaic ring chromosome 21 remained undetected. Depending on input DNA quality, the average read length ranged from 8.3-17.6 kb and the coverage was >20x for all samples.De novoassembly resulted in a limited number of contigs per individual (N50 range 6-73 Mb) clearly separating the two alleles in most cases, enabling direct characterization of the chromosomal rearrangements.ConclusionsWe have within a national pilot study successfully demonstrated the utility of HiFi Revio lrGS as a clinical analysis of chromosomal rearrangements.