Abstract
BackgroundDespite extensive efforts devoted to predicting protein-coding genes in genome sequences, many bona fide genes have not been found and many existing gene models are not accurate in all sequenced eukaryote genomes. This situation is partly explained by the fact that gene prediction programs have been developed based on our incomplete understanding of gene feature information such as splicing and promoter characteristics. Additionally, full-length cDNAs of many genes and their isoforms are hard to obtain due to their low level or rare expression. In order to obtain full-length sequences of all protein-coding genes, alternative approaches are required.ResultsIn this project, we have developed a method of reconstructing full-length cDNA sequences based on short expressed sequence tags which is called sequence tag-based amplification of cDNA ends (STACE). Expressed tags are used as anchors for retrieving full-length transcripts in two rounds of PCR amplification. We have demonstrated the application of STACE in reconstructing full-length cDNA sequences using expressed tags mined in an array of serial analysis of gene expression (SAGE) of C. elegans cDNA libraries. We have successfully applied STACE to recover sequence information for 12 genes, for two of which we found isoforms. STACE was used to successfully recover full-length cDNA sequences for seven of these genes.ConclusionsThe STACE method can be used to effectively reconstruct full-length cDNA sequences of genes that are under-represented in cDNA sequencing projects and have been missed by existing gene prediction methods, but their existence has been suggested by short sequence tags such as SAGE tags.
Highlights
Despite extensive efforts devoted to predicting protein-coding genes in genome sequences, many bona fide genes have not been found and many existing gene models are not accurate in all sequenced eukaryote genomes
While serial analysis of gene expression (SAGE) tags that correspond to existing gene models can be used to evaluate the abundance of gene expression, there are a large number of SAGE tags that do not correspond to existing gene models
Tag based reconstruction of full-length cDNA sequence of novel genes Expressed sequence tags that cannot be aligned to the C. elegans virtual transcriptome suggest the existence of yet unannotated genes [13,21]
Summary
Despite extensive efforts devoted to predicting protein-coding genes in genome sequences, many bona fide genes have not been found and many existing gene models are not accurate in all sequenced eukaryote genomes. The C. elegans gene set is still far from complete for the following reasons: First, because Genefinder, like other gene prediction programs, was developed based on an incomplete understanding of gene structures, it suffers from both false. In this project, we explored how to reconstruct fulllength gene models for genes that are not correctly represented in the current gene set, using expressed sequence tags obtained in large-scale gene expression projects. These SAGE tags suggest the existence of additional coding exons, splice variants [20], or novel genes
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