Development and Optimalisation of Selective Whole Genome Amplification to Enrich Samples with Low Toxoplasma Gondii DNA Copies for Whole Genome Sequencing

Abstract

Toxoplasma gondii (T. gondii) is a protozoan parasite that can cause severe disease in immunocompromised individuals, including pregnant women and foetuses. Understanding the genome variation of T. gondii at the population level is crucial for identifying virulence factors and designing effective therapies. However, obtaining sufficient quantities of highquality parasite DNA from clinical samples for obtaining whole genome sequencing (WGS) data is challenging due to the overwhelming presence of host DNA. In this study, selective whole genome amplification (SWGA) was applied to efficiently enrich the genomic DNA of T. gondii from a high background of human (host) DNA. T. gondii DNA was successfully amplified and sequenced from T. gondii samples cultured in human foreskin fibroblasts cells with low parasitaemia using specifically designed primer sets, without the need for host DNA depletion. In order to obtain WGS data from low parasitaemia samples, SWGA primer sets were designed to preferentially bind to T. gondii genomic DNA in samples that were predominantly composed of human host DNA. In the study multiple sets of primers were designed and the efficiency of two SWGA primers sets to amplify and enrich the target T. gondii DNA from a human DNA background confirmed by real-time quantitative polymerase chain reaction (qPCR). In order to further test the effectiveness of SWGA to selectively enrich T. gondii DNA, the specificity of the selected primer sets were analysed, and the reaction conditions were optimised, and the limit of detection of target DNA determined. Additionally, the influence of SWGA reaction additives, such as bovine serum albumin (BSA), was also tested to obtain the highest yield possible. The optimised SWGA reactions were applied to DNA extracted from Tg_RH and Tg_PRU strains of T. gondii grown in human cell cultures using two sets of SWGA primers (set-1 and set_2) and the pre-SWGA and post-SWGA DNA sequenced, and genome coverage analysed. The study provides important insights into the coverage depth and percentage coverage across the T. gondii genome. The average coverage depth for pre-SWGA samples Tg_RH strain and TG_PRU strain was found to be 67X and 21X of the T. gondii reference genome, respectively. However, the post-SWGA samples had a higher average coverage depth, 243X and 163X for the Tg_RH and Tg_PRU strains for primer set_1, and 179X and 104X respectively for primer set_2. It was also observed that the average coverage depth of human genomic DNA decreased for all post-SWGA samples when mapped to the human reference genome. The study findings suggest that SWGA technology is an effective method for obtaining high-quality T. gondii DNA from clinical samples, and the technique has the potential for further applications in understanding the genome variation of T. gondii at the population level. Furthermore, the SWGA method was used to evaluate the feasibility of amplifying T. gondii DNA from low-level positive pork samples with varying T. gondii genome copy numbers. The results demonstrated the SWGA method's effectiveness in amplifying T. gondii DNA in two out of five samples. The use of the SWGA method resulted in a significant increase in coverage of T. gondii reference genome, particularly in one sample. These findings suggest that the SWGA method has the potential to be used for the detection and amplification of T. gondii in food samples. However, the low success rate of amplification in this study highlights the need for further optimisation of the SWGA method, such as designing primers specific for the enrichment of T. gondii parasite from pig genome background.

Date of Award	2024
Original language	English
Awarding Institution	Aberystwyth University
Supervisor	Justin Pachebat (Supervisor) & Martin Swain (Supervisor)