Genetic analysis using long-read sequencing to overcome the difficulties in <i>VWF</i> gene

<h4>Background</h4>Genetic defects in von Willebrand factor (VWF) can lead to von Willebrand disease (VWD). Identifying causative or modifier variants of <i>VWF</i> is crucial for the diagnosis, classification, and clinical management of VWF disorders. However, owing to the length (178 kb) and complexity of <i>VWF</i> and the presence of the pseudogene <i>VWFP1</i>, Sanger sequencing or short-read next-generation sequencing is often challenging.<h4>Objectives</h4>This study aimed to establish a long-read sequencing method using Oxford nanopore technology (ONT) to overcome difficulties associated with <i>VWF</i> gene analysis.<h4>Methods</h4>Genetic analyses were established using genomic DNA from a healthy donor and validated using 3 VWF disorder patient samples. Long-range (∼15 kb) polymerase chain reaction was optimized to obtain 21 amplicons covering the entire <i>VWF</i> gene, avoiding unwanted amplification due to repetitive sequences and <i>VWFP1</i>. ONT nanopore sequencing data were analyzed using software programs, including Clair3, Longshot, and Sniffles. The identified candidate variants were verified by several approaches such as Sanger sequencing and haplotyping.<h4>Results</h4>The entire <i>VWF</i> gene was successfully read using ONT nanopore sequencing, with >200 variants called in each patient sample. A rare missense variant, p.(Gln2442His) and a rare 2599 bp deletion were identified in patients 2 and 3, respectively. However, the deletion was confirmed as long-range polymerase chain reaction artifacts, which warrant attention when using this method.<h4>Conclusion</h4>This study presents an optimal solution using ONT nanopore sequencing to identify variants in <i>VWF</i>, which may improve the diagnosis of VWF disorders.