The future of preimplantation genetic testing

Preimplantation genetic testing (PGT), where embryos are screened for chromosomal abnormalities prior to implantation, is commonly applied to enhance the success of in vitro fertilisation (IVF). Balanced reciprocal translocations, which describes the exchange of genetic material between two nonhomologous chromosomes, are one of the most common chromosomal abnormalities in humans, with an estimated incidence of 0.25%. However, this class of aberration cannot be easily identified using existing analysis techniques, such as microarray and short-read sequencing, that rely on detecting copy number changes.

Based on the facility of nanopore technology to generate long sequencing reads that allow delineation of large and complex structural variants, researchers at The University Hospital of Hong Kong assessed its potential to support the identification of balanced translocations in embryo samples.

‘…instead of spending days preparing the NGS library, it takes only 90 min for library preparation of nanopore sequencing’ 1

In this study, genomic DNA from nine known balanced translocation carriers was prepared for sequencing using the Ligation Sequencing Kit prior to running on the MinION device. Between approximately 8 Gb and 19 Gb of data were generated per sample, with the mean sequencing depth of the translocated chromosomes ranging from 2.5-fold to 6.2-fold. Using NanoSV, all known translocation breakpoints were accurately detected for each carrier, as confirmed by Sanger sequencing of the breakpoint flanking regions. The genomic context of each breakpoint was also identified,

‘Breakpoint mapping by nanopore sequencing offers a number of advantages compared with other methods, such as SNP haplotyping and NGS’ 1

revealing eight gene interruptions, six breakpoints located within repeat elements, one breakpoint located within a segmental duplication, and a number of microdeletions (Figure 10).

The team then developed PCR primers for each breakpoint, which they used in a retrospective analysis of embryo samples conceived by the carriers. This technique was shown to enable the accurate determination of translocation carrier status for the embryos.

‘Long reads are superior to short reads regarding detection of complex chromosome rearrangements’ 2

Figure 1: Low coverage nanopore whole-genome sequencing enabled accurate breakpoint mapping of a balanced translocation and associated deletions between chromosomes 8 (a) and (10 (b), and subsequent design of breakpoint PCR primers (c & d) for sample 100649. Red arrow indicates breakpoints predicted by NanoSV. N: noncarrier; C: translocation carrier; B: negative control; M: DNA molecular weight marker; der: derivative chromosome. Figure from Chow et al. MethodsX. 6:2499-2503 (2019). Figure available under Creative Commons license (

The application of nanopore sequencing to identify balanced translocations has also been demonstrated by Zhang et al 2, who used a similar low-coverage nanopore sequencing strategy to delineate breakpoints in carrier samples, which included a complex deletion-inversion-deletion event that was missed using traditional short-read sequencing. The nanopore data was used to inform subsequent chromosomal screening of embryos using a SNPbased microarray platform. According to the researchers, ‘nanopore long-read sequencing is a powerful method to assay chromosomal inversions and identify exact break points’ 2.

Researchers at Columbia University Medical Center have also published data demonstrating how nanopore sequencing may, in the future, support the rapid screening of embryos for chromosomal aneuploidy 3. In contrast to existing assays, which take in excess of 12 hours, nanopore sequencing results were obtained within just a few hours of sample receipt — a timeline potentially amenable to same-day embryo transfer.

‘Compared to traditional NGS technologies, nanopore-based sequencing has the distinct advantage of sequencing 15,000 times faster, delivering sequencing results in real-time and a 99x lower capital equipment cost’ 3

1. Chow, J.F.C., Cheng, H.H.Y., Lau, E.Y.L, Yeung, W.S.B., and Ng, E.H.Y. Distinguishing between carrier and noncarrier embryos with the use of long-read sequencing in preimplantation genetic testing for reciprocal translocations. Genomics 112(1):494–500 (2020).

2. Zhang, S. et al. Long-read sequencing and haplotype linkage analysis enabled preimplantation genetic testing for patients carrying pathogenic inversions. J. Med. Genet. 56(11):741–749 (2019).

3. Wei, S., Weiss, Z.R., Gaur, P., Forman, E. and Williams, Z. Rapid preimplantation genetic screening using a handheld, nanopore-based DNA sequencer. Fertility and Sterility 110(5):910-916 (2018).