Nanopore sequencing for detecting reciprocal translocation carrier status in preimplantation genetic testing

Abstract

Background Balanced reciprocal translocation (BRT) is one of the most common chromosomal abnormalities that causes infertility, recurrent miscarriage, and birth defects. Preimplantation genetic testing (PGT) is widely used to select euploid embryos for BRT carriers to increase the chance of a healthy live birth. Several strategies can be used to distinguish reciprocal translocation carrier embryos from those with a normal karyotype; however, these techniques are time-consuming and difficult to implement in clinical laboratories. In this study, nanopore sequencing was performed in two reciprocal translocation carriers, and the results were validated using the next-generation sequencing-based method named, “Mapping Allele with Resolved Carrier Status” (MaReCs). Results The translocation breakpoints in both reciprocal translocation carriers were accurately identified by nanopore sequencing and were in accordance with the results obtained using MaReCs. More than one euploid non-balanced translocation carrier embryo was identified in both patients. Amniocentesis results revealed normal karyotypes, consistent with the findings by MaReCs and nanopore sequencing. Conclusion Our results suggest that nanopore sequencing is a powerful strategy for accurately distinguishing non-translocation embryos from translocation carrier embryos and precisely localizing translocation breakpoints, which is essential for PGT and aids in reducing the propagation of reciprocal translocation in the population.

Nanopore sequencing with T2T‐CHM13 for accurate detection and preventing the transmission of structural rearrangements in highly repetitive heterochromatin regions in human embryos

“…To verify the feasibility of T2T‐CHM13 for balanced translocation, we performed a retrospective analysis using patient 2 as the subject 11 . We had already identified the precise breakpoints of 26 208 295 (13q11) and 33 942 281 (17q11.2) using GRCh37.…”

“…To verify the feasibility of T2T‐CHM13 for balanced translocation, we performed a retrospective analysis using patient 2 as the subject. 11 We had already identified the precise breakpoints of 26 208 295 (13q11) and 33 942 281 (17q11.2) using GRCh37. However, TGS revealed that the actual location of the breakpoint was some distance from the centromere, which was not entirely in line with the karyotype.…”

Clinical-grade whole genome sequencing-based haplarithmisis enables all forms of preimplantation genetic testing

“…We demonstrate the ability to detect structural rearrangements and distinguish normal from balanced embryos through both a direct approach, leveraging paired-end sequencing information from the embryo samples, and an indirect approach, which involves analyzing flanking haplotypes. Even though various strategies exist to differentiate normal from balanced embryos, including MaReCs 42 , a method based on shallow sequencing 43 , and a method based on Nanopore sequencing technology 44 , they cannot be integrated with other workflows for other PGT purposes as each workflow requires different technologies. Finally, we show the comparability and reproducibility of heteroplasmy levels between traditional day-3 blastomere biopsy with PCR-RFLP and our WGS-PGT method.…”

Preimplantation genetic testing: A remarkable history of pioneering, technical challenges, innovations, and ethical considerations

“…TGS has been applied successfully for PGT. Applications have included testing for aneuploidies and structural rearrangement (Tan et al, 2023; Xia et al, 2023). This includes the detection of balanced reciprocal translocations, and identification of breakpoints to within 1 bp (Xia et al, 2023).…”

“…Applications have included testing for aneuploidies and structural rearrangement (Tan et al, 2023; Xia et al, 2023). This includes the detection of balanced reciprocal translocations, and identification of breakpoints to within 1 bp (Xia et al, 2023). In one study, TGS could be performed with fewer reads, and concordance between NGS and TGS was nearly 99% for aneuploidies of 10 Mb or greater, with somewhat better performance of TGS for defects between 5 and 10 Mb, but diminished efficiency for both platforms for detecting defects of <10 Mb, and NGS appeared to perform better for lower quality samples (Tan et al, 2023).…”