Selective Transfer of Euploid Noncarrier Embryos with the Use of Long-Read Sequencing in Preimplantation Genetic Testing for Reciprocal Translocation

Abstract

Introduction: Balanced reciprocal translocation is one of the most common chromosomal abnormalities in humans. Carriers are usually phenotypically normal but are at an increased risk of infertility, recurrent miscarriage or having affected children. Preimplantation genetic testing for chromosomal structural rearrangement (PGT-SR) offers a way to screen against embryos with unbalanced translocation during in vitro fertilization (IVF) treatment cycles. In this case report, we demonstrated a new approch to discriminate carrier from noncarrier embryos. First, high-resolution breakpoint mapping was performed using long-read sequencing, followed by breakpoint PCR. In the treatment cycle, PGT-SR was performed by NGS and euploid noncarrier embryos were selected using breakpoint PCR.

Material & methods: a) Long-read sequencing A couple requested PGT-SR because the wife, aged 33 years, was known to be a carrier of balanced translocation [46,XX,t(7:13)(p13;q12.3)]. A sequencing library (1D) was prepared on the genomic DNA of the wife using the SQK-LSK-108 kit (Oxford Nanopore, UK) according to the manufacturers's protocol. 1D sequencing was performed on a MinION flow cell (R9.4, Oxford Nanopore) with a 48-hour protocol on MinKNOW (2.2.12). Local base calling was performed by Albacore 2.3.1. After sequence alignemnt, breakpoints were determined by NanoSV 1.2.0. b) Breakpoint PCR Based on the predicted breakpoints that correlated with cytogenetic report, breakpoint PCR primers were designed and validated using genomic DNA of couple and the sequences flanking the breakpoints were confirmed by Sanger sequencing. c) PGT-SR Whole genomic amplification (WGA, REPLI-g Single Cell kit) was performed followed by PGT-SR by NGS (VeriSeq-PGS, Illumina). Breakpoint PCR primers validated in (b) were used to discriminate carrier from noncarrier embryos.

Results: Nanopore sequencing obtained an average read length of 7000 bases. The total no. of based aligned to human genome was 16 Giga bases. Breakpoints were accurately predicted on chromosomes 7 and 13. Eighteen oocytes were retrieved and 15 were fertilized. Trophectoderm biopsy was performed on 9 blastocyts. After PGT-SR, there were 2 euploid embryos and 2 mosaic embryos. Four embryos were unbalanced carriers and one embryo with no result due to failure in WGA. Breakpoint PCR was used to discriminate carrier from noncarrier embryos. Both euploid embryos and one mosaic embryo were noncarriers while the other mosaic embryo was a carrier. One euploid noncarrier blastocytst was replaced in FET cycle. The patient is pregnant and has been referred to prenatal clinic for follow up (10-week at the time of abstract submission).

Conclusion: Long-read sequencing enables accurate high-resolution breakpoint mapping directly on balanced reciprocal translocation carriers. In addition, nanopore sequencing required simple library preparation (90 minutes) and low capital cost, therefore the cost of preclincial test (nanopore sequencing and validation of breakpoint PCR) for the first PGT-SR cycle is approximately US$ 1000. In conclusion, we demonstrated a new method to discriminate carrier from noncarrier embryos with the use of nanopore sequencing and breakpoint PCR. This method allows patients to prioritize the transfer of euploid noncarrier embryos.