Long-read sequencing on the SMRT platform enables efficient haplotype linkage analysis in preimplantation genetic testing for β-thalassemia

Purpose This study aimed to evaluate the value of long-read sequencing for preimplantation haplotype linkage analysis. Methods The genetic material of the three β-thalassemia mutation carrier couples was sequenced using single-molecule real-time sequencing in the 7.7-kb region of the HBB gene and a 7.4-kb region that partially overlapped with it to detect the presence of 17 common HBB gene mutations in the Chinese population and the haplotypes formed by the continuous array of single-nucleotide polymorphisms linked to these mutations. By using the same method to analyze multiple displacement amplification products of embryos from three families and comparing the results with those of the parents, it could be revealed whether the embryos carry disease-causing mutations without the need for a proband. Results The HBB gene mutations of the three couples were accurately detected, and the haplotype linked to the pathogenic site was successfully obtained without the need for a proband. A total of 68.75% (22/32) of embryos from the three families successfully underwent haplotype linkage analysis, and the results were consistent with the results of NGS-based mutation site detection. Conclusion This study supports long-read sequencing as a potential tool for preimplantation haplotype linkage analysis.