A novel single tube amplification and barcoding approach for preimplantation genetic testing (PGT-A) for aneuploidy detectionusing Next Generation Sequencing

Abstract

Introduction There are several methods to prepare embryo biopsy samples for Next Generation Sequencing (NGS) for Preimplantation Genetic Testing for Aneuploidy (PGT-A). DOPlify® provides a flexible technology to not only amplify whole genomes and target sequences using RHS’ target sequence enrichment (TSE) protocol, but also provides a mechanism to incorporate platform specific sequences for NGS. The use of PCR barcoding during whole genome amplification provides several laboratory efficiencies compared to sequential whole genome amplification followed by library preparation methods, including the reduction of hands on time, total protocol time and reagent requirements for sample preparation, all of which are equally relevant across small clinics through to high throughput service laboratories. Here we describe the development of a novel approach which allows PCR barcoding of PGT-A samples in a single tube using PCR that has been developed on multi- cell samples of known ploidy as a model of trophectoderm biopsy. Material and Methods Five-cell samples manually sorted from aneuploid cell lines (Coriell Institute) were prepared for sequencing using a two-stage, single tube protocol. DNA was amplified using standard DOPlify® kit reagents (RHS Ltd) then Ion Torrent (ThermoFisher) NGS adapter sequences and barcodes were subsequently incorporated utilising a second PCR step within the same tube. Incorporation of the adapter sequences at both the 5’ and 3’ ends of the amplified DNA was quantified using qPCR (Kapa Biosystems) with adapter sequence-specific primers (RHS Ltd). The barcoded samples were pooled and sequenced. The sequencing data was bioinformatically aligned to hg19, sequencing metrics collated and the data analysed to determine sample ploidy status. Results More than 8 different barcoding methods were trialled and 3 were successful. Measurable differences were observed for WGA DNA yield, DNA fragment size range and the 5’ and 3’ adapter sequence incorporation ratio (1:1-1:5). For the successful protocols, concordance of the sample karyotype was achieved for all cell lines sequenced (n=5 for each method). The most time efficient protocol produced amplified, sequencing ready samples within a total time of 3 hrs 30 min with a hands on time of about 30 mins, with further reduction of total protocol time to less than 3 hrs currently being evaluated. Additional time improvements may come from automation. Conclusions Leveraging the unique DOPlify® technology, this novel method provides a single tube amplification and barcoding protocol to allow rapid, scalable and economical sequencing of embryo biopsy samples for PGT-A using next generation sequencing. With the possibility of incorporating RHS’ target sequence enrichment (TSE) protocol, this novel PCR barcoding approach will then allow combined PGT-M and PGT-A.