[
International Worm Meeting,
2003]
As part of a consortium of laboratories in Canada and Sweden (see McKay, et al, "Gene expression profiles in cells, tissues and development of C. elegans"), we have developed computational and biological approaches to assist in high throughput analysis of gene expression. Biological source material for SAGE libraries was generated using synchronized populations as well as micro-dissected gut tissue. We are also refining GFP-mediated cell sorting techniques to isolate other cell types from disrupted embryos. SAGE data are generated and tracked in an automated pipeline from sequencing of cloned concatamers through to annotation, quality control and mapping of individual tags. Tags are associated with attributes such as cumulative phred scores, source clone, parent ditag and tag-to-gene mapping data to aid in downstream quality filtering and analysis steps. To facilitate tag-to-gene mapping and comparison of SAGE and Affymetrix GeneChip technologies, a virtual C. elegans transcriptome with both confirmed and estimated untranslated regions (UTRs) for all known and predicted transcripts was constructed using the current gene models and expressed sequence tag (EST) data annotated in wormbase. We are also refining tag-to-gene mapping methods in order to detect previously undocumented transcripts and alternative splice variants. Comparison of the Affymetrix C. elegans chip with the current virtual transcriptome showed that about 97% of the confirmed or predicted transcripts are detected by all or part of at least one probe set. At the time of writing, four developmentally staged SAGE libraries to be used in developmental gene expression profiling have been completed. Preliminary SAGE studies and results of a direct comparison of gene expression levels measured from the same biological source material with SAGE, long-SAGE and Affymetrix GeneChip technologies will be presented. This project is funded by Genome British Columbia and Genome Canada.