Application of the third generation sequencing in preimplantation genetic testing

Abstract

Preimplantation genetic testing (PGT) refers to the testing methods for evaluation of the genetic make-up of in-vitro cultured embryos before transfer into the uterus in assisted reproduction programs. Carriers of translocations are at a high risk of infertility, recurrent miscarriage, or having an affected pregnancy. In the ideal case, selective transfer of an euploid noncarrier embryo prevents the patients from physical and psychological trauma associated with chromosomal abnormalities on one hand and eliminates the inheritance of such chromosomal abnormalities in the family on the other. The hypothesis of the project is that the long read length uniquely offered by third generation sequencing on Oxford Nanopore Technologies platform (thereafter, nanopore sequencing) facilitates the reads to cover across the regions of rearrangements, copy number variants (CNVs), and microdeletions / microduplications (MMs). This allows the high resolution mapping of regions of interest and enables the development of specific PCR for carrier diagnosis and direct mutation analyses for PGT. Next generation sequencing (NGS) offers a way to screen out embryos with whole chromosome aneuploidies but with variable resolution for segmental imbalances. The resolution limit of the NGS platform for PGT-SR was determined and a general criterion on assessing patients requiring PGT-SR was established. It was also demonstrated that the translocation breakpoints could be accurately mapped in base-pair resolution by using the nanopore sequencing. This enabled the design of breakpoint PCR for determination of the carrier status of embryos. Furthermore, in cases of Robertsonian translocation where nanopore sequencing was not applicable, alternative strategy by haplotyping with universal short tandem repeat (STR) markers panels was developed to determine the carrier status of embryos. Application of nanopore sequencing was extended to PGT for monogenic diseases (PGT-M). Comprehensive genetic testing for pathogenic CNVs usually offers limited resolution to exon level, therefore PGT-M for CNVs mainly relies on indirect haplotype analysis. However, haplotype analysis is not feasible in cases with germline mosaicism, de novo mutations or when an affected family member is not available for phasing. Mapping of pathogenic CNVs by nanopore sequencing was established which facilitated integration of direct mutation detection testing with haplotype analysis for development of more accurate PGT-M assays. Mapping of microdeletions / microduplications (MMs) by nanopore sequencing was also investigated. It was found that, breakpoints of non-recurrent MMs but not of recurrent MMs, can be characterized by nanopore sequencing. PGT on recurrent MMs by targeted PCR was feasible, while STR haplotyping strategy was applied for PGT of recurrent MMs. In conclusion, nanopore sequencing was applied as a comprehensive strategy for pre-PGT workup for couples requesting PGT-SR or PGT-M. In a single nanopore sequencing run, information on mapping of breakpoints / CNVs can be quickly gathered for establishing PGT protocol. Nanopore sequencing also allowed PGT-A / PGT-SR and high resolution mapping of translocation breakpoints directly on embryo biopsies. Universal STR panels was established to determine the carrier status of embryo for Robertsonian translocation carriers.