Combined B-thalassemia mutation detection and PGT-A using a novel PCR barcoding approach for ion torrent NGS

Abstract

Thalassemia was the 4th most frequent monogenic disorder screened using Preimplantation Genetic Testing (PGT-M) according to 10 years of ESHRE data collection. The ability to combine Thalassemia-specific PCR primers and Whole Genome Amplification (WGA) for concurrent PGT-M and PGT for aneuploidy (PGT-A) together in a single PCR reaction represents a unique approach to maximise the screening opportunity for a single embryo biopsy. DOPlify provides a flexible technology to amplify whole genomes and clinically relevant sequences using RHS’ Target Sequence Enrichment (TSE) protocol and also a unique mechanism to incorporate specific sequences needed for Next Generation Sequencing (NGS) sample preparation, offering a streamlined solution for combined PGT-M and PGT-A. The aim of this study was to develop a novel approach that allows amplification and PCR barcoding of samples for combined PGT-A and PGT-M for β-Thalassemia mutation testing in a single tube for Ion Torrent (Thermo Fisher) NGS. 5-cell samples were manually sorted from euploid and aneuploid cell lines (Coriell Institute) and prepared for sequencing using a two-stage, single tube protocol. DNA was amplified using standard DOPlify kit reagents with a modified primer (RHS Ltd). The TSE protocol (RHS Ltd) was used with the inclusion of Haemoglobin subunit beta (HBB)-specific PCR primers in the same reaction. Enrichment of HBB sequence during DOPlify WGA with TSE was confirmed using HBB-specific PCR. Sequencing was performed by pooling the HBB PCR products with the DOPlify with TSE products. Publicly available Ion Torrent NGS adapter sequences and barcodes were subsequently incorporated utilising a second PCR step within the same WGA PCR tube. Sequencing was performed and the data was bioinformatically aligned to hg19, analysed to determine sample ploidy and viewed using Integrative Genomics Viewer (Broad Institute). Accurate detection of 7-32Mb sub-chromosomal duplications and deletions, and whole chromosome aneuploidies concordant with the expected karyotype of the cell lines was achieved. Target sequence enrichment using HBB-specific PCR primers for β-Thalassemia mutation detection was also successful. Breadth of coverage of 100% and read depths in excess of 150x for HBB were achieved when HBB PCR products were pooled back into DOPlify with TSE products at a 1:10 ratio. This contrasts with incomplete coverage and read depths of approximately 13x for DOPlify with TSE, and an absence of mapped reads for control DOPlify only. Leveraging the unique characteristics of DOPlify, this novel method provides a single tube amplification and barcoding protocol to allow rapid and economical sequencing of embryo biopsy samples for PGT-A. Together with the targeted and concurrent enrichment of specific clinically relevant sequences during WGA, a suitable depth of coverage for PGT-M was also achieved.