Philosophiae Doctor - PhD (Biotechnology)

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https://hdl.handle.net/10566/13468

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Browsing by Author "Christoffels, Alan"

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    A computational framework for transcriptome assembly and annotation in non-model organisms: the case of venturia inaequalis
    (University of the Western Cape, 2014) Kimbung, Stanley Mbandi; Christoffels, Alan; Jasper, D; Rees, G
    In this dissertation three computational approaches are presented that enable optimization of reference-free transcriptome reconstruction. The first addresses the selection of bona fide reconstructed transcribed fragments (transfrags) from de novo transcriptome assemblies and annotation with a multiple domain co-occurrence framework. We showed that selected transfrags are functionally relevant and represented over 94% of the information derived from annotation by transference. The second approach relates to quality score based RNA-seq sub-sampling and the description of a novel sequence similarity-derived metric for quality assessment of de novo transcriptome assemblies. A detail systematic analysis of the side effects induced by quality score based trimming and or filtering on artefact removal and transcriptome quality is describe. Aggressive trimming produced incomplete reconstructed and missing transfrags. This approach was applied in generating an optimal transcriptome assembly for a South African isolate of V. inaequalis. The third approach deals with the computational partitioning of transfrags assembled from RNA-Seq of mixed host and pathogen reads. We used this strategy to correct a publicly available transcriptome assembly for V. inaequalis (Indian isolate). We binned 50% of the latter to Apple transfrags and identified putative immunity transcript models. Comparative transcriptomic analysis between fungi transfrags from the Indian and South African isolates reveal effectors or transcripts that may be expressed in planta upon morphogenic differentiation. These studies have successfully identified V. inaequalis specific transfrags that can facilitate gene discovery. The unique access to an in-house draft genome assembly allowed us to provide preliminary description of genes that are implicated in pathogenesis. Gene prediction with bona fide transfrags produced 11,692 protein-coding genes. We identified two hydrophobin-like genes and six accessory genes of the melanin biosynthetic pathway that are implicated in the invasive action of the appressorium. The cazyome reveals an impressive repertoire of carbohydrate degrading enzymes and carbohydrate-binding modules amongst which are six polysaccharide lyases, and the largest number of carbohydrate esterases (twenty-eight) known in any fungus sequenced to date
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    Genomic and proteomic analysis of drought tolerance in Sorghum (Sorghum bicolor (L.) Moench)
    (University of the Western Cape, 2014) Woldesemayat, Adunga,Abdi; Christoffels, Alan; Ndimba, Bongani.K
    Drought is the most complex phenomenon that remained to be a potential and historic challenge to human welfare. It affects plant productivity by eliciting perturbations related to a pathway that controls a normal, functionally intact biological process of the plant. Sorghum (Sorghum bicolor (L.) Moench), a drought adapted model cereal grass is a potential target in the modem agricultural research towards understanding the molecular and cellular basis of drought tolerance. This study reports on the genomic and proteomic findings of drought tolerance in sorghum combining the results from in silica and experimental analysis. Pipeline that includes mapping expression data from 92 normalized cDNAs to genomic loci were used to identify drought tolerant genes. Integrative analysis was carried out using sequence similarity search, metabolic pathway, gene expression profiling and orthology relation to investigate genes of interest. Gene structure prediction was conducted using combination of ab initio and extrinsic evidence-driven information employing multi-criteria sources to improve accuracy. Gene ontology was used to cross-validate and to functionally assign and enrich genes. An integrated approach that subtly combines functional ontology based semantic data with expression profiling and biological networks was employed to analyse gene association with plant phenotypes and to identify and genetically dissect complex drought tolerance in sorghum. The gramene database was used to identify genes with direct or indirect association to drought related ontology terms in sorghum. Where direct association for sorghum genes were not available, genes were captured using Ensemble Biomart by transitive association based on the putative functions of sorghum orthologs in closely related species. Ontology mapping represented a direct or transitive association of genes to multiple drought related ontology terms based on sorghum specific genes or orthologs in related species. Correlation of genes to enriched gene ontology (GO)-terms (p-value < 0.05) related to the whole-plant structure was used to determine the extent of gene-phynotype association across-species and environmental stresses.
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    A transcriptome analysis of apple (Malus x domestica Borkh.) cv ‘golden delicious’ fruit during fruit growth and development
    (2013) Chikwambi, Zedias; Rees, D.J.G.; Christoffels, Alan
    The growth and development of apple (Malus x domestica Borkh.) fruit occurs over a period of about 150 days after anthesis to full ripeness. During this period morphological and physiological changes occur defining fruit quality. These changes are a result of spatial and temporal patterns of gene expression during fruit development as regulated by environmental, genetic and environmental-by-genetic factors. A number of previous studies partially characterised the transcriptomes of apple leaf, fruit pulp, whole fruit, and peel plus pulp tissues, using cDNA micro arrays and other PCR based technologies. These studies, however, remain limited in throughput and specificity for transcripts of low abundance. Hence, the aim of this project was to apply a high throughput technique to characterise the full mRNA transcriptome of the ‘Golden Delicious’ fruit peels and pulp tissues in order to understand the molecular mechanisms underlying the morphophysiological changes that occur during fruit development.