High_throughput_phenotyping_using_parallel_sequencing_of_RNA_interference_targets_in_the_African_trypanosome-sc-2010-12-09
High_throughput_phenotyping_using_parallel_sequencing_of_RNA_interference_targets_in_the_African_trypanosome
RnD T. Brucei RNAi study. An expression vector, containing random fragments of the Brucei genome was used was used to generate RNAis, and the project uses a TraDIS-like library preparation protocol to identify these genomic fragments in the surviving organisms.
Abstract: African trypanosomes are major pathogens of humans and livestock and represent a model for studies of unusual protozoal biology. We describe a high throughput phenotyping approach termed RNA interference (RNAi) target sequencing, or RIT-seq that, using Illumina sequencing, maps fitness-costs associated with RNAi. We scored the abundance of >90,000/ /integrated RNAi targets recovered from trypanosome libraries before and after induction of RNAi. Data are presented for 7,435 protein coding sequences, >99% of the non-redundant set in the /Trypanosoma brucei/ genome. Analysis of two major life cycle stages and differentiated libraries revealed genome  scale knockdown profiles of growth and development, linking thousands of previously uncharacterised genes to essential functions. Genes underlying the essential functions of trypanosome biology are highlighted, including the emphasis on constitutive  post-transcriptional gene expression control; the importance of compartmentalised glycolysis and flagellar motility in the bloodstream and of carboxylic acid metabolism and phosphorylation during differentiation. The current data-set provides functional information for several thousand 'hypothetical' genes and much needed genetic  validation to identify new drug-targets. RIT-seq represents a versatile new tool for genome-scale functional analyses and for the exploitation of genome sequence data.