Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

High throughput decoding of anti trypanosomal drug efficacy and resistance

ABSTRACT: We used all five current human African trypanosomiasis drugs for genome-scale RNA interference (RNAi) target sequencing (RIT-seq) screens in Trypanosoma brucei, revealing the transporters, organelles, enzymes and metabolic pathways that function to facilitate anti-trypanosomal drug action. RIT-seq profiling links more than fifty additional genes to drug action. This data has been described in the following article [doi:10.1038/nature10771] and its further analysis can be freely submitted for publication. For information on the proper use of data shared by the Wellcome Trust Sanger Institute (including information on acknowledgement), please see http://www.sanger.ac.uk/datasharing/. Protocol - Assembly of the bloodstream-form T. brucei RNAi library and the RNAi target sequencing (RIT-seq) approach were reported previously (Alsford, S. et al. High-throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome. Genome Res 21, 915-924, 264 doi:gr.115089.110 [pii] 265 10.1101/gr.115089.110 (2011). Briefly, a tetracycline-inducible RNAi plasmid library, containing randomly sheared genomic fragments (mean ~600 bp) under the control of head-to-head T7 promoters, was targeted to a single genomic locus that had been validated for robust expression. For this study, the library was grown under inducing conditions with drug selection and genomic DNA was isolated from surviving populations. For chemical RIT-seq profiling, adapter-ligated sequencing libraries were prepared from each genomic DNA sample and used to amplify DNA fragments containing RNAi cassette-insert junctions in semi-specific PCR reactions; one primer was specific for the RNAi vector and the other for the Illumina adapter. Size-selected DNA was sequenced with 76 cycle paired-end runs on an Illumina GAII. Illumina sequencing reads containing a nine-base RNAi vector junction sequence were then mapped to the T. brucei reference genome. Where loss of function increases drug tolerance, RNAi-target sequence representation is increased relative to the otherwise susceptible population, revealing hot spots.

INSTRUMENT(S): Illumina Genome Analyzer II

ORGANISM(S): Trypanosoma brucei

SUBMITTER:

PROVIDER: E-ERAD-30 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

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

Alsford Sam S Turner Daniel J DJ Obado Samson O SO Sanchez-Flores Alejandro A Glover Lucy L Berriman Matthew M Hertz-Fowler Christiane C Horn David D

Genome research 20110301 6

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, ...[more]

PMID: 21363968

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Project description:The protozoan parasites Trypanosoma brucei spp. are responsible for important human and livestock diseases in sub Saharan Africa. In the mammalian blood, two developmental forms of the parasite exist: proliferative ?slender? forms and transmissible ?stumpy? forms that are quiescent, awaiting uptake in a tsetse fly bloodmeal. The slender to stumpy differentiation is a density-dependent response that resembles quorum sensing in microbial systems and is crucial for the parasite life cycle, ensuring both infection chronicity and disease transmission. The response is triggered by an elusive ?stumpy induction factor? (SIF) whose intracellular signaling pathway is also completely uncharacterized. Laboratory-adapted (monomorphic) trypanosome strains cannot respond to SIF, but can generate forms with stumpy characteristics when exposed to cell permeable cAMP and AMP analogues. Exploiting this, we have used a genome-wide RNAi library screen to identify the signaling components driving stumpy formation. In separate screens, monomorphic parasites were exposed to cell permeable cAMP or AMP analogues to select cells that remained proliferative and so were unresponsive to these signals. Genome-wide ion torrent-based RNA interference Target sequencing (RIT-seq) identified a cohort of genes implicated in all steps of the signaling pathway, from purine metabolism, through signal transducers (kinases, phosphatases) to gene expression regulators. The identified genes at each step have been validated in cells naturally capable of stumpy formation, confirming their role in SIF-induced density sensing and cellular quiescence. RNA was isolated from AnTat1.1 cells grown in mice with or without doxycycline. RBP7 (Tb927.10.12100) RNAi lines were grown in mice with or without doxycycline. Since the RBP7 RNAi is leaky (six independent cell lines were analysed and all were significantly leaky), transcripts that showed elevated or reduced abundance in both RNAi induced and uninduced populations were identified in comparison to the AnTat1.1 control. A similar approach was adopted for the RBP7 over-expressing lines.

Dataset Information

High throughput decoding of anti trypanosomal drug efficacy and resistance

Publications

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

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