Transcriptome analysis for Caenorhabditis elegans based on novel expressed sequence tags

Abstract

Background: We have applied a high-throughput pyrosequencing technology for transcriptome profiling of Caenorhabditis elegans in its first larval stage. Using this approach, we have generated a large amount of data for expressed sequence tags, which provides an opportunity for the discovery of putative novel transcripts and alternative splice variants that could be developmentally specific to the first larval stage. This work also demonstrates 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 worms utilizing high-throughput sequencing technology. We have shown that approximately 14% of the newly sequenced expressed sequence tags map completely or partially to genomic regions where there are no annotated genes or splice variants and therefore, imply that these are novel genetic structures. Expressed sequence tags, which map to intergenic (around 1000) and intronic regions (around 580), may represent novel transcribed regions, such as unannotated or unrecognized small protein-coding or non-protein-coding genes or splice variants. Expressed sequence 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 extension of the coding or untranslated regions. We have also discovered that intergenic and intronic expressed sequence tags, 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 stage worms, in order to predict novel transcripts that might be specifically or predominantly expressed in the first larval stage.

Conclusion: We have demonstrated the use of a high-throughput sequencing methodology to efficiently produce a snap-shot of transcriptional activities occurring in the first larval stage of C. elegans development. Such application of this new sequencing technique allows for high-throughput, genome-wide experimental verification of 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.

Figure 1

454 ESTs mapping to Caenorhabditis elegans transcripts. (A) Histogram showing the distribution of 454 expressed sequence tags (ESTs) mapping to Caenorhabditis elegans transcripts. Coordinate 0 on the x-axis represents the 5'-end of the transcripts. (B) Summary of 454 EST mapping result to the C. elegans transcriptome and genome. (C) Categorization of genomic 454 EST hits.

Figure 2

The intergenic region on chromosomes with unique 454 expressed sequence tags. (A) The intergenic region on chromosome III with 49 unique 454 expressed sequence tags (ESTs). (B) The intergenic region on chromosome I with the most number of unique 454 ESTs (99) in this analysis. (C) The intergenic region on chromosome II with 62 unique 454 ESTs. The 454 EST clusters in the middle of these intergenic regions with black vertical bars represent deep EST coverage, and conservation of these regions between Caenorhabditis elegans and C. briggsae is shown. These ESTs may represent a novel gene or extension of the neighboring gene. Note that the genomic regions shown are not to the same scale.

Figure 3

Examples of intronic and gapped expressed sequence tags. (A) An example of intronic expressed sequence tags (ESTs) showing 454 ESTs mapped to the gene, K09E2.3, which is added to a recent WormBase release (WS180) within the intron of K09E2.2. There are also other ESTs recently added that confirm K09E2.3. (B) An example of a gapped-EST suggesting alternative splicing or correction of the current gene model. Note that the genomic regions shown are not to the same scale.

Figure 4

Comparative analyses of 3' splice sites, GC contents, and cross-species sequence conservation. (A) The conservation of consensus 3'-end splice sites (TTTTCAG) of confirmed transcripts and transcripts with exon-intron boundary 454 expressed sequence tag hits. (B) A comparison of 454 expressed sequence tags and Caenorhabditis elegans whole genome for guanine-cytosine content of different genomic regions. (C) Average ClustalW alignment score comparisons for intergenic regions with or without 454 expressed sequence tags for different numbers of orthologous sequences. (D) Chart showing 3524 unique Caenorhabditis elegans intergenic 454 expressed sequence tags mapped to C. briggsae, C. remanei, and C. brenneri.

Figure 5

Examples of 454 ESTs mapped to known or predicted ncRNAs. (A), (B) Representative 454 expressed sequence tag data, which identify known non-coding RNAs. (C) The most conserved cross-species intronic 454 expressed sequence tags hit mapping to a RNAz non-coding RNA prediction. Note that the genomic regions shown are not to the same scale.