Application of whole genome sequencing and metagenomics for diagnosis of tuberculosis

Abstract

Globally, tuberculosis kills more people than any other infectious disease. Control of the epidemic is impeded by poor diagnostic approaches. My original contribution to knowledge presented in this thesis is towards diagnosis of tuberculosis by (meta)genomic approaches. In the work presented here, I established proof-of-principle that tuberculosis can be detected, identified and somewhat characterised using a shotgun metagenomics approach. I developed an approach for DNA extraction directly from sputum followed by metagenomic sequencing that allowed me to detect sequences from the M. tuberculosis complex in all sixteen samples with low coverage of the H37Rv reference genome. This allowed me to assign the lineage of the MTBC species in thirteen of these samples. This was the basis of the first publication to sequencing tuberculosis without prior culture. I determined that the proportion of human reads in the resulting metagenomic data was a major limitation to characterising the MTBC organisms with greater resolution and sought, though unsuccessfully, to determine methods to remedy this. In doing so, I identified a number of considerations that need to be made when designing studies of human DNA depletion from such heterogeneous clinical samples in the future. Addressing some of the other limitations to using genomics in the diagnosis of tuberculosis, I studied the genotype-phenotype association of first-line drug resistance found in patients in Peru and evaluated the performance diagnostic approaches used. This identified novel mutations associated with pyrazinamide resistance, flaws in the MODS method of antibiotic resistance testing and variation in resistance prediction tools