Long-read NGS guided preimplantation genetic testing for chromosomal structural rearrangement

"What can short-read sequencing do?" This question opened Chris's talk, as he discussed how array CGH and short-read sequencing have been applied to PGT in embryonic biopsies. These techniques have been used to profile "single cells" from peripheral blood and amniotic fluid, and have identified, for example, chromosomal gain and loss of copy number. Chris also highlighted that such a short-read sequencing approach has been used for resolving aberrant karyotypes.

However, Chris questioned, is short-read sequencing enough? He explained how he has used the Oxford Nanopore MinION platform in his research, giving the real-life example of applying nanopore sequencing in the context of ectrodactyly (also called the lobster hands phenotype). From long-read whole-genome nanopore sequencing of an IVF-PGT patient with ectrodactyly, obtaining over 50% of reads with a length ≥ 31 kbp, a duplication breakpoint was revealed in a 36 kbp sequencing read; PCR and Sanger sequencing validated this unique duplication breakpoint. From this result, IVF embryos were then screened for the presence of the breakpoint, and the patient was able to decide about embryo implantation.

Chris finally described how he has performed Cas9 enrichment to enrich for copy number variation sequences of interest, prior to long-read nanopore sequencing.

To conclude, Chris stated that long-read nanopore sequencing can be used to successfully identify structural rearrangement breakpoints in blood cells, and can indirectly improve the accuracy and efficiency of PGT, compared to array CGH and short-read sequencing. Long-repeat elements associated with DiGeorge and Robersonian translocations may still require alternative approaches. He stated that the Cas9 enrichment protocol could be successfully used, instead of whole-genome sequencing, to identify structural rearrangements, with the benefit of saving time.