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Laboratorio di Genomica Funzionale
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As deep sequencing has rapidly gained popularity for transcriptome analysis because of its ability to generate digital and quantitative information on annotated genes, we are implementing mRNA-Seq for expression analysis, using Vitis vinifera as model organism. The cDNAs libraries obtained by retrotrascription of fragmented mRNA, are sequenced with an Illumina Genome Analyzer IIx. By using two different softwares for alignment of sequences on the reference genomes (ELAND and BOWTIE) the short reads (36 or 75 nucleotides, single reads or paired ends) are mapped to exons, introns or intergenic regions, hence identifying new exons or new genes not predicted. We have adapted modules of the ERANGE program for digital gene expression analysis, detection of alternative splicing (annotated or new) and SNPs discovery. A strong bioinformatics engagement was necessary to develop a modified module in ERANGE software dedicated to the alternative splicing detection. Expression values, calculated as reads per kilobase of exon model per million mapped reads (RPKM) are then used to monitor changes in gene expression. We are currently evaluating several statistical approaches to identify genes which are significantly modulated, and we are comparing their output with the results obtained on the same reference material with different microarray designs
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Genetics & Heredity - Genetics & Heredity
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pezzotti
univr
