Transcriptome Analysis for Caenorhabditis elegans Based on Novel Expressed Sequence Tags

Background: We have applied a high-throughput pyrosequencing technology for transcriptome profiling ofCaenorhabditis elegans in its first larval stage. Using this approach, we have generated a large amount of data forexpressed sequence tags, which provides an opportunity for the discovery of putative novel transcripts andalternative splice variants that could be developmentally specific to the first larval stage. This work alsodemonstrates the successful and efficient application of a next generation sequencing methodology.Results: We have generated over 30 million bases of novel expressed sequence tags from first larval stage wormsutilizing high-throughput sequencing technology. We have shown that approximately 14% of the newly sequencedexpressed sequence tags map completely or partially to genomic regions where there are no annotated genes orsplice variants and therefore, imply that these are novel genetic structures. Expressed sequence tags, which mapto intergenic (around 1000) and intronic regions (around 580), may represent novel transcribed regions, such asunannotated or unrecognized small protein-coding or non-protein-coding genes or splice variants. Expressedsequence tags, which map across intron-exon boundaries (around 300), indicate possible alternative splice sites,while expressed sequence tags, which map near the ends of known transcripts (around 600), suggest extensionof the coding or untranslated regions. We have also discovered that intergenic and intronic expressed sequencetags, which are well conserved across different nematode species, are likely to represent non-coding RNAs.Lastly, we have incorporated available serial analysis of gene expression data generated from first larval stageworms, in order to predict novel transcripts that might be specifically or predominantly expressed in the firstlarval stage.Conclusion: We have demonstrated the use of a high-throughput sequencing methodology to efficientlyproduce a snap-shot of transcriptional activities occurring in the first larval stage of C. elegans development. Suchapplication of this new sequencing technique allows for high-throughput, genome-wide experimental verificationof known and novel transcripts. This study provides a more complete C. elegans transcriptome profile and,furthermore, gives insight into the evolutionary and biological complexity of this organism.