Project description:We have developed a method based on 454 sequencing of 3â?? 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 â??virtual transcriptomeâ?? provides extensive coverage depth, and can be used for the set 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. The microarray designed on the berriesâ?? transcriptome derived from half of a 454 run detected the expression of 19,609 genes, and proven 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. Microarray oligo probes were designed based on 454 sequencing of 3'-ends of transcripts of a sample constituted by pooling RNAs from grape berries at 6 time points from veraison to whitering. The array was used to analyze the same pool and results were compared to GrapeArray 1.2 developed by the Italian-French Public Consortium for Grapevine Genome Characterization.
Project description: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 set 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. The microarray designed on the berries’ transcriptome derived from half of a 454 run detected the expression of 19,609 genes, and proven 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.