P-721 Nanopore sequencing for detecting reciprocal translocation carrier status in preimplantation genetic testing
Authors: Yao Z 
Published: June 2023

Abstract

Study question
Analyze the concordance between nanopore sequencing and MaReCs to validate the feasibility of nanopore sequencing in distinguishing normal embryos from carrier embryos in PGT-SR cycles.

Summary answer
Nanopore sequencing is a powerful strategy for accurately distinguishing nontranslocation embryos from translocation carrier embryos and precisely localizing translocation breakpoints.

What is known already
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.

Study design, size, duration
The Nanopore Sequencing was performed on two pregnant patients conducted on Mapping Allele with Resolved Carrier Status” (MaReCs) in PGT-SR cycles to identify translocation breakpoints for a year. Combined conventional MaReCs ’results, amniocentesis was performed to identify karyotypes at 18–20 weeks of gestation to verify the correctness of the Nanopore Sequencing.

Participants/materials, setting, methods
A total of 11 embryos from 2 couple1 and 12 embryos from couple 2 were tested simultaneously in PGT-SR cycles with a combination strategy of mate pair sequencing and PCR breakpoint analysis, SNP array-based comprehensive chromosome screening (CCS), NGS following micro-dissecting junction region (Micro-Seq) to distinguish between normal and carrier embryos. The Nanopore Sequencing based on TGS was performed on 2 couples. Lastly, amniocentesis was performed to test karyotypes.

Main results and the role of chance
The translocation breakpoints in both reciprocal translocation carriers were accurately identifed 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.

Limitations, reasons for caution
One limitation is that it is not possible to identify the translocation breakpoints of Robertsonian translocation carriers or those located in the gap regions of the human genome. In addition, high-molecular-weight genomic DNA and large amounts of peripheral blood samples would be a challenge most PGT centers.

Wider implications of the findings
The nanopore sequencing accurately detects translocation breakpoints in BRT carriers and distinguishes translocation-free embryos from balanced diploid embryos in clinical PGT-SR cycles. It is superior to short reads (NGS) for the detection of translocation breakpoints, which shows broad prospects for clinical applications in blocking translocation propagation in the population.

Trial registration number
Not applicable