Using full genome sequence to compare pathogenic and non-pathogenic isolates of Theileria orientalis in Australia
- Publication Type:
- Thesis
- Issue Date:
- 2020
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ππ©π¦πͺππ¦π³πͺπ’ π°π³πͺπ¦π―π΅π’ππͺπ΄ is an apicomplexan haemoparasite that causes oriental theileriosis in cattle. The parasite is an economic burden globally and there are currently no suitable therapies for the disease in Australia. It is vital to understand the genome of π. π°π³πͺπ¦π―π΅π’ππͺπ΄ to aid the development of efficacious vaccines and/or therapeutics. Next generation sequencing provides an opportunity to comparatively study the π. π°π³πͺπ¦π―π΅π’ππͺπ΄ genome to elucidate its diversity and population structure.
Removal of contaminating host DNA is critical for next-generation sequencing (NGS) of intracellular parasites. This thesis investigated the efficacy of π. π°π³πͺπ¦π―π΅π’ππͺπ΄ DNA enrichment on fresh and frozen clinical blood samples using cellulose leukocyte depletion columns, a commercial microbiome enrichment kit, and selective whole genome amplification (SWGA). All methods were demonstrated to be effective but with unique limitations. qPCR assays were developed to estimate host-to-parasite ratio and identified the commercial microbiome kit to be the most feasible enrichment method for this project.
Long read sequencing of three common π. π°π³πͺπ¦π―π΅π’ππͺπ΄ genotypes was achieved with an Oxford Nanopore MinION flow-cell. Using four distinct assembly methods and manual scaffolding, chromosomal-level assemblies of all three genomes was achieved. Genome annotation and comparative analysis revealed species-level, structural and gene content differences between pathogenic (genotype Ikeda, str. Robertson) and non-pathogenic (genotypes Chitose and Buffeli, str. Fish Creek and str. Goon Nure, respectively) strains similar to that of transforming Theilerias, π. π±π’π³π·π’ and π. π’π―π―πΆππ’π΅π’.
To generate high-quality SNPs, a Parasite Variant Pipeline (PVP) was developed with stringent quality filters to eliminate low coverage, erroneous or non-coding SNPs. Validation was performed with 270 simulated sequences over three levels of coverage breadths and real-world data. To account for errors caused by large indel events, PVP was revalidated and validation metrics revealed no false positives as well as > 88% PVP sensitivity over 16 Γ coverage.
Finally, a study of diversity and recombination within clinical Australian isolates was conducted with methods and tools investigated in the preceding research chapters. Illumina sequencing was performed on clinical samples that were enriched for parasite DNA. Sequencing data was processed through PVP with long read sequenced and previously published reference sequences to detect high-quality SNPs that were used to identify signatures of meiotic recombination and genetic diversity with genomic distance measures. A total of 15,901 high-quality SNPs were detected by PVP and results indicate π. π°π³πͺπ¦π―π΅π’ππͺπ΄ has low sequence diversity in Australia, which suggests potential for the development of a successful vaccine.
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