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An approach to determine the Transcriptome of T. b. rhodesiense from Sleeping Sickness patients
Human African Trypanosomiasis (HAT) is a neglected tropical disease that mainly affects the poorest people in sub Saharan Africa. HAT is caused by two subspecies of Trypanosoma brucei; T. b. rhodesiense is found in Eastern Africa and causes the acute form of the disease, while T. b. gambiense is found in Western Africa and causes the chronic form of the disease. With no reliable diagnostic screening test, available drugs being rather toxic, and emerging cases of drug resistant strains, research on the molecular aspects of the trypanosome is being carried out with the hope of identifying potential drug targets and diagnostic markers. Since most studies are carried out on cultured blood stream trypanosomes, the extent to which these parasites are representative of a real human infection is not known. Therefore the aim of this study was to analyze the transcriptome of clinical isolates of T. b. rhodesiense from patient peripheral blood and cerebral spinal fluid by high throughput sequencing. But given the low parasitaemia during active infection, I developed a splice leader priming based Polymerase chain reaction method to specifically amplify nanogram amounts of trypanosome total RNA in microgram amounts of Human cellular RNA, to an amount sufficient for sequencing. The amplification method resulted in trypanosome transcripts covering 60% of the T. brucei 6772 unique genes, and with an expression threshold of 5 Rpkm. The sequenced amplified libraries (four replicates) were highly reproducible and comparable to unamplified libraries generated in the same way. However a comparison to the conventional RNASeq generated library showed distortions in the transcriptome, which could be corrected for and used to analyze clinical samples. An analysis of methods used to purify trypanosomes from blood showed that; even though DEAE chromatography and reticulocyte lysis resulted in 10 times more parasites than those in the buffy coat isolation method, reticulocyte lysis resulted in distortion of the transcriptome. However the DEAE chromatography trypanosomes transcriptome which was more comparable to the buffy coat, was not highly reproducible. The analysis of genomes from the trypanosomes isolated from the patients showed a level of heterogeneity between the samples with significant gene copy number variations observed mainly in the multi copy genes.
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570 Biowissenschaften, Biologie
570 Life sciences