A novel targeted long-read sequencing-based preimplantation genetic testing method for α-thalassemia (tlrPGT-α-thal)

<h4>Purpose</h4>The purpose is to develop a preimplantation genetic testing (PGT) method for α-thalassemia in one reaction, without requiring a proband, using targeted long-range polymerase chain reaction (LR-PCR) followed by long-read sequencing (LRS).<h4>Methods</h4>We developed a targeted LRS-based PGT method for α-thalassemia (tlrPGT-α-thal). This novel approach detects both HBA-associated variants and informative linked fragments in peripheral regions by targeted LR-PCR in one reaction.<h4>Results</h4>A total of 115 samples, including 10 couples and 95 embryos, were subjected to this method. Available heterozygous SNPs were used to distinguish four parental haplotypes. Haplotype linkage analysis was successfully performed in all families, and the results of embryos were all consistent with control results obtained through conventional methods (next-generation sequencing, Sanger sequencing and gap-PCR). This confirmed that tlrPGT-α-thal achieved 100% sensitivity and accuracy.<h4>Conclusion</h4>This study has demonstrated the accuracy and clinical utility of tlrPGT-α-thal. This method holds significant promise for clinical application for patients requiring PGT for α-thalassemia. Furthermore, the methodology could potentially be extended to PGT for monogenic disorders (PGT-M) in other diseases.