High-throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome

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 associa...

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Bibliographic Details
Published in:Genome research 2011-06, Vol.21 (6), p.915-924
Main Authors: Alsford, Sam, Turner, Daniel J, Obado, Samson O, Sanchez-Flores, Alejandro, Glover, Lucy, Berriman, Matthew, Hertz-Fowler, Christiane, Horn, David
Format: Article
Language:English
Subjects:
Computational Biology
DNA Primers
DNA Primers - genetics
Gene Library
Genetic Fitness
Genetic Fitness - genetics
Genome, Protozoan
Genome, Protozoan - genetics
Genomics
Genomics - methods
High-Throughput Nucleotide Sequencing
High-Throughput Nucleotide Sequencing - methods
Life Sciences
Method
Phenotype
Plasmids
Plasmids - genetics
RNA Interference
Sequence Analysis, DNA
Sequence Analysis, DNA - methods
Trypanosoma brucei
Trypanosoma brucei brucei
Trypanosoma brucei brucei - genetics
Trypanosoma brucei brucei - physiology
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Summary: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.
ISSN:1088-9051
1549-5469
DOI:10.1101/gr.115089.110