title: High-throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome.
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 7435 protein coding sequences, >99% of a non-redundant set in the Trypanosoma brucei genome. Analysis of bloodstream and insect life-cycle stages and differentiated libraries revealed genome-scale knockdown profiles of growth and development, linking thousands of previously uncharacterized and "hypothetical" genes to essential functions. Genes underlying prominent features of trypanosome biology are highlighted, including the constitutive emphasis on post-transcriptional gene expression control, the importance of flagellar motility and glycolysis in the bloodstream, and of carboxylic acid metabolism and phosphorylation during differentiation from the bloodstream to the insect stage. The current data set also provides 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.
authors.affiliation: London School of Hygiene & Tropical Medicine, London WC1E 7HT, United Kingdom.
authors.forename: Sam
authors.initials: S
authors.lastname: Alsford
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Turner
authors.forename: Samson O
authors.initials: SO
authors.lastname: Obado
authors.forename: Alejandro
authors.initials: A
authors.lastname: Sanchez-Flores
authors.forename: Lucy
authors.initials: L
authors.lastname: Glover
authors.forename: Matthew
authors.initials: M
authors.lastname: Berriman
authors.forename: Christiane
authors.initials: C
authors.lastname: Hertz-Fowler
authors.forename: David
authors.initials: D
authors.lastname: Horn
mesh_terms: D019295:Computational Biology; D017931:DNA Primers / Q000235:genetics; D015723:Gene Library; D056084:Genetic Fitness / Q000235:genetics; D018503:Genome, Protozoan / Q000235:genetics*; D023281:Genomics / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D034622:RNA Interference; D017422:Sequence Analysis, DNA / Q000379:methods*; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000502:physiology
chemical_list: D017931:DNA Primers