We have developed a method based on 454 sequencing of 3’ cDNA fragments from a normalized library constructed from pooled RNAs to generate, through de novo reads assembly, a large catalog of unique transcripts in organisms for which a comprehensive collection of transcripts or the complete genome sequence, is not available. This “virtual transcriptome” provides extensive coverage depth, and can be used for the setting up of a comprehensive microarray based expression analysis. We evaluated the potential of this approach by monitoring gene expression during berry maturation in Vitis vinifera as if no other sequence information was available for this species. The microarray designed on the berries’ transcriptome derived from half of a 454 run detected the expression of 19,609 genes, and proved to be more informative than one of the most comprehensive grape microarrays available to date, the GrapeArray 1.2 developed by the Italian-French Public Consortium for Grapevine Genome Characterization, which could detect the expression of 15,556 genes. Therefore we showed that this approach provides a powerful tool to rapidly build up an extensive catalog of unique transcripts that can be successfully used to develop a microarray for large scale analysis of gene expression in any species, without the need for prior sequence knowledge.

Combining next-generation pyrosequencing with microarray for large scale expression analysis in non-model species

BELLIN, Diana;DELLEDONNE, Massimo
2009

Abstract

We have developed a method based on 454 sequencing of 3’ cDNA fragments from a normalized library constructed from pooled RNAs to generate, through de novo reads assembly, a large catalog of unique transcripts in organisms for which a comprehensive collection of transcripts or the complete genome sequence, is not available. This “virtual transcriptome” provides extensive coverage depth, and can be used for the setting up of a comprehensive microarray based expression analysis. We evaluated the potential of this approach by monitoring gene expression during berry maturation in Vitis vinifera as if no other sequence information was available for this species. The microarray designed on the berries’ transcriptome derived from half of a 454 run detected the expression of 19,609 genes, and proved to be more informative than one of the most comprehensive grape microarrays available to date, the GrapeArray 1.2 developed by the Italian-French Public Consortium for Grapevine Genome Characterization, which could detect the expression of 15,556 genes. Therefore we showed that this approach provides a powerful tool to rapidly build up an extensive catalog of unique transcripts that can be successfully used to develop a microarray for large scale analysis of gene expression in any species, without the need for prior sequence knowledge.
9788890062292
sequencing; model plants; transcrptomics
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11562/478793
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