Genomic and transcriptomic analysis of Mycobacterum bovis using next generation sequencing technologies

Mycobacterium bovis is the causative agent of bovine tuberculosis and is endemic in Ireland. Illumina platforms are the industry gold standard for whole genome sequencing (WGS) applications, including epidemiology of M. bovis. The lag time between M. bovis culture DNA extraction, library preparation, and genome sequencing is a significant barrier in the contemporaneous attribution of infection in cattle and wildlife in the Republic of Ireland. Third generation sequencing (TGS), such as Oxford Nanopore Technology (ONT), are increasingly applied to the same genomic epidemiological workflows. However WGS modalities work with different physical and chemical technologies, leading to differing per- base accuracy, length of resulting reads, differences in the platforms’ size and portability, and sample batch numbers. Furthermore, long reads may be more easily assembled de novo, assisting in accurate assessment of large structural variation and species identification of highly repetitive and GC-rich M. bovis genomes. The aims of this thesis were to compare and contrast the NGS Illumina platform to the TGS Nanopore platform. In doing so, the sensitivity of each for genomic epidemiology purposes could be measured. Additionally, short and long reads produced by the respective workflows could be combined to define the structural variation of a novel M. bovis isolate, TB18-003488. Finally, transcriptomic analysis of TB18-003488 was used to reveal any variation present in its gene expression profile as compared to the well annotated reference M. bovis AF2122/97. The results of this thesis demonstrate that Illumina and Nanopore workflows can be used for the same epidemiological workflows, producing similar results. These results also indicate that there is a degree of systematic bias present in the data produced by the two platforms, although work will be required to assess this in greater detail. Additionally, a result of this thesis is a draft genome for M. bovis TB18-003488 that defines and annotates the consequence of a large genomic region absent in TB18-003488 that resembles the RD1 regions of M. bovis BCG and Mycobacterium microti. Transcriptomic analysis of TB18-003488 revealed alteration in transcription of genes involved in lipid metabolism, dormancy, and transcriptional regulators, compared to the AF2122/97 reference. The implications of these findings are discussed.