Cost-effective and flexible preimplantation genetic testing (PGT) by nanopore adaptive sampling

Genetic diseases encompass a spectrum of disorders resulting from DNA variations. Preimplantation genetic testing (PGT) is a critical strategy for preventing recurrent miscarriage, fetal 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. In contrast, 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 non-targeted regions, offering a more cost-effective and flexible alternative. In this proof-of-principle study, we employed low-coverage short-read next-generation sequencing (NGS), microarray analysis and nanopore adaptive sampling to analyze 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. Nanopore adaptive sampling 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 nanopore adaptive sampling in assisted reproduction. Compared with traditional PGT techniques based on low-coverage short-read NGS combined with microarray analysis, our work highlights that nanopore adaptive sampling is a promising tool for PGT, offering cost-effective solutions, especially for incomplete pedigrees and de novo mutations, and provides preliminary proof-of-principle evidence for its broader clinical application.