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. 2006 Aug 3:7:196.
doi: 10.1186/1471-2164-7-196.

Generation of a large scale repertoire of Expressed Sequence Tags (ESTs) from normalised rainbow trout cDNA libraries

Marina Govoroun  1 , Florence Le Gac, Yann Guiguen
Affiliations

Generation of a large scale repertoire of Expressed Sequence Tags (ESTs) from normalised rainbow trout cDNA libraries

Marina Govoroun et al. BMC Genomics. .

Abstract

Background: Within the framework of a genomics project on livestock species (AGENAE), we initiated a high-throughput DNA sequencing program of Expressed Sequence Tags (ESTs) in rainbow trout, Oncorhynchus mykiss.

Results: We constructed three cDNA libraries including one highly complex pooled-tissue library. These libraries were normalized and subtracted to reduce clone redundancy. ESTs sequences were produced, and 96,472 ESTs corresponding to high quality sequence reads were released on the international database, currently representing 42.5% of the overall sequence knowledge in this species. All these EST sequences and other publicly available ESTs in rainbow trout have been included on a publicly available Website (SIGENAE) and have been clustered into a total of 52,930 clusters of putative transcripts groups, including 24,616 singletons. 57.1% of these 52,930 clusters are represented by at least one Agenae EST and 14,343 clusters (27.1%) are only composed by Agenae ESTs. Sequence analysis also reveals that normalization and especially subtraction were effective in decreasing redundancy, and that the pooled-tissue library was representative of the initial tissue complexity.

Conclusion: Due to present work on the construction of rainbow trout normalized cDNA libraries and their extensive sequencing, along with other large scale sequencing programs, rainbow trout is now one of the major fish models in term of EST sequences available in a public database, just after Zebrafish, Danio rerio. This information is now used for the selection of a non redundant set of clones for producing DNA micro-arrays in order to examine global gene expression.

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Figures

Figure 1
Figure 1
Percentage of novel EST clusters found as a function of the number of clones sequenced in the rainbow trout pooled-tissue cDNA library. N = Normalization. S1 = First subtraction. S2 = Second subtraction.
Figure 2
Figure 2
Histogram of cluster sizes. Repartition in the different cluster size classes of the complete collection of trout clusters (black squares) and of Agenae specific clusters (open squares), both expressed in proportion of all clusters, based on Sigenae rainbow trout EST clustering version 3. The table represented the number of clusters in each cluster size class.
Figure 3
Figure 3
Diagram showing the number and the relative proportion (%) of shared and unique clusters. Shared clusters contain ESTs from different projects (AGENAE, USDA, Others) and unique clusters only contain ESTs originating from one project. Clusters are made of one (called singletons and represented under brackets) or more ESTs assembled together following clustering analysis. USDA libraries were 01 to 10RT – NCCCWA libraries and 115RT – NCCCWA library.
See this image and copyright information in PMC

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