Optimisation of whole genome amplification specifically for non-invasive PGT-A using spent embryo culture media

Abstract

Introduction The clinical use of non-invasive preimplantation genetic testing for aneuploidy (PGT-A) requires concordance of the spent embryo culture media result to embryo biopsy result and the ability to distinguish maternal contamination from the embryonic DNA, especially for a euploid female result. Although concordance of spent embryo culture media and trophectoderm biopsy has been reported at as high as 95% following the collection of samples at day 5-7 using DOPlify™ ( Lane et al, 2017 ), the ability to test media collected earlier in culture requires an increased level of sensitivity. Additionally, there are a number of known PCR inhibitors in culture media, including salts and proteins, which need to be overcome. Here we describe acustomised whole genome amplification (WGA) protocol designed specifically for the amplification of DNA contained in spent embryo culture media. Material and Methods Spent embryo culture media was collected by clinics with ethics approval from single embryo culture droplets and pooled prior to storage at -20°C. Pooling was used to provide a consistent test sample to analyse across a range of protocol variants to overcome sample to sample variability. To accommodate the use of a larger sample input volume into the WGA and the composition of culture media post embryo culture, samples were amplified using a re-formulated single step version of DOPlify™. The single step protocol removes the DOPlify™ lysis step and requires the addition of only one reagent mastermix into the PCR tube containing the culture media, making this protocol ideal for automation. The same pooled media samples were amplified in parallel using the standard DOPlify™ protocol. WGA DNA yield was assessed following gel electrophoresis and HS Qubit quantification (ThermoFisher) and sequenced on a MiSeq sequencer (Illumina) according to the standard PG-Seq™ 48 sample protocol. The sequencing data for each sample was bioinformatically aligned to hg19 and the sequencing QC metrics for each protocol were collated and compared. Results A total of 32 protocol variants were tested in triplicate. Outputs measured were yield, size distribution of PCR amplicons, %GC content and PG-Seq™ NGS software quality scores. With the optimal protocol, WGA DNA yield per sample increased on average 5-fold following the amplification of pooled media samples using a re-formulated version of DOPlify™ with output measures comparable to biopsy results. It is also possible to detect mitochondrial DNA from the amplified spent culture media, although with fewer reads per sample than biopsies provide. The same increased yield and acceptable QC results have since been obtained for individual culture droplets from embryos cultured in 10-60ul of media from a range of manufacturers. Conclusions Successful non-invasive PGT-A using spent embryo culture media requires a customised approach to amplify DNA found within this complex sample type. Accurate and representative amplification of the embryo DNA is essential for the success of this non-invasive approach. Further incorporation of RHS’ target sequence enrichment protocol would provide a mechanism to detect maternal DNA within a spent culture media sample.