Cost-effective and Flexible Preimplantation Genetic Testing (PGT) Using Adaptive Sampling-based Targeted Nanopore Sequencing (ASTN-seq)

Genetic diseases encompass a spectrum of disorders resulting from DNA variations. Preimplantation genetic testing (PGT) is a critical strategy for preventing recurrent miscarriage, foetal malformations, and the birth of children affected by chromosomal abnormalities and monogenic disorders. Traditional PGT techniques necessitate comprehensive pedigree genetic data for haplotype linkage analysis, whereas PGT employing third-generation sequencing (TGS) has distinct advantages, particularly in cases of incomplete pedigree information, de novo mutations, and complex pathogenic variants. Nevertheless, the widespread application of TGS-based PGT in clinical practice encounters hurdles owing to its high costs. Targeted sequencing technologies present a promising solution by selectively enriching regions of interest while disregarding nontargeted areas, offering a more cost-effective and flexible alternative. In this study, we employed next-generation sequencing (NGS) and adaptive sampling-based targeted nanopore sequencing (ASTN-seq) to analyse samples from five couples who carried balanced translocations and HBB gene pathogenic mutations, as well as three additional couples with monogenic diseases caused by mutations in PKD1, ASNS, or ALPL. ASTN-seq successfully identified various mutations and facilitated haplotype linkage analysis, confirming its accuracy and reliability. Successful embryo transfer and subsequent prenatal diagnosis in certain families underscore the potential of ASTN-seq in assisted reproduction. Compared with traditional NGS-based PGT techniques, our work highlights that ASTN-seq is a promising tool for PGT, offering cost-effective solutions for PGT, especially for incomplete pedigrees and de novo mutations.