EST arraying
This project is an adjunct to the Schistosoma genome project, and will involve arraying of ESTs.
Collaborators
- Prof. R Alan Wilson
- Dr Peter D. Ashton
- Dr Rachel E. Adamson
- Ms Rachel S Curwen Dept of Biology, Univ of York
Main staff involved
- Al Ivens Project Manager
- Theresa Feltwell Snr Tech. Ass.
- Nefeli Nikolaidou-Katsaridou Snr Tech. Ass.
Outline of Project
The lung stage of the human blood fluke Schistosoma mansoni is a validated target of protective immunity in rodents and primates, but sequences from this stage are very underrepresented in the public domain. This project comprises two phases:
- sequencing of ESTs
- arraying of appropriate ESTs for profiling studies
Phase One
At the outset of the project, two novel cDNA libraries from the lung stage had been constructed (see details below). Sequencing data have been generated (~5300 reads, out of a total of ~10,000) from these libraries).
Phase Two
The data obtained suggested that the Incyte library had the highest quality, but was inappropriate for arraying. New cEST libraries are in the process of being made, and will be arrayed subject to QC.
The sequence data from this important stage of the parasite life-cycle will underpin complementary studies of the schistosome proteome also being undertaken at the University of York. Alan Wilson's group at the University of York have established MALDI-TOF and tandem MS proteomics techniques for S. mansoni and have been funded by the UK BBSRC to use these techniques to investigate larval development and sexual maturation. York University is currently constructing a major proteomics facility with UK government and Wellcome Trust funding which will be available for later stages of this project.
Lung stage cDNA libraries
- custom-made by Incyte Genomics, this library is 5'-enriched but not directionally cloned. Analysis of 177 pilot sequences has revealed 66% novel transcripts, and SignalP/PSort searching indicates that 10% encode signal peptides.
- made by Phil Conde from York in Prof. Beck's laboratory in Giessen University, is currently being characterised. It has large inserts and high primary complexity (>500,000 individual clones). Most sequences to date are novel and redundancy is on a reasonable scale. A subtracted version is being made using cDNA from day 20 larvae to enrich for stage-specific transcripts.
Other Schistosoma arrays
WHO have funded the production of nylon membrane arrays containing a non-redundant set of 4,500 S. mansoni and S. japonicum EST sequences (PI David Johnston, Natural History Museum, London, daj@nhm.ac.uk). This project is ongoing, but has taken far longer than originally anticipated due to both logistical problems and clone supply problems. A pilot batch of glass arrays containing some 500 EST sequences has also been generated in collaboration with Karl Hoffman, Pathology Department, University of Cambridge and an initial evaluation of these arrays clearly demonstrates the potential of this resource. It is anticipated that the full arrays will now be made available on both glass and nylon. Dedicated, 500 clone S. japonicum- glass arrays will also be produced.
