Genetic analysis using long-read sequencing to overcome the difficulties in gene.

BACKGROUND

Genetic defects in von Willebrand factor (VWF) can lead to von Willebrand disease (VWD). Identifying causative or modifier variants of is crucial for the diagnosis, classification, and clinical management of VWF disorders. However, owing to the length (178 kb) and complexity of and the presence of the pseudogene , Sanger sequencing or short-read next-generation sequencing is often challenging.

OBJECTIVES

This study aimed to establish a long-read sequencing method using Oxford nanopore technology (ONT) to overcome difficulties associated with gene analysis.

METHODS

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 gene, avoiding unwanted amplification due to repetitive sequences and . 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.

RESULTS

The entire 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.

CONCLUSION

This study presents an optimal solution using ONT nanopore sequencing to identify variants in , which may improve the diagnosis of VWF disorders.