se discovery rates than when using statistical t-tests. Because RPKM values are determined on basis of 1000 nt, a decrease in contig size will result in increased noise. In order to visualize noise, MA plots were generated to display the ratio M = log2 vs. the average expression by RPKM A = +log2)/2 of the contigs as grouped in various sizeclasses. Iterative Homology Search and Annotation FASTA consensus sequences of the assembled contigs were annotated using an iterative homology search approach consisting of consecutive BLAST steps against different databases. After each BLAST step, XML results were analyzed with the Blast2GO pipeline using default settings and the best BLAST hit was recovered. Those 10069503 contigs without or with uninformative descriptions were submitted to the next BLAST step; the others were considered to be well annotated and were kept. The different BLAST steps performed were 1) nucleotide BLAST against EST salmonid sequences of the SIGENAE database, the Information System of the AGENAE program of INRA with updated annotation; 2) BLASTx against the zebrafish Reference Sequence protein database and 3) BLASTx against the refSeq Metazoa protein database excluding D. rerio: The query `srcdb_refseq AND Metazoa NOT ��D. rerio’��was used in NCBI to retrieve a ��gi��list of non-D. rerio refSeqs and create a restricted alias of the refseq_protein database containing the refSeqs from all metazoa except D. rerio. Results from this step were not AZD 2171 filtered for uninformative descriptions and they were all added to the final annotation file. were selected with annotations that indicated key muscle functions. For validation by Q-PCR, 10 contigs for white muscle and 11 contigs for red muscle were selected. The contig sequences associated with the genes guanylate-binding protein and troponin T3b skeletal fast isoform 1 were similar for red muscle and white muscle. Primers were designed within the overlapping region of the two sequences for red muscle and white muscle and the same 16388798 primers were used for both tissues. Other contig sequences were different for both tissues but were associated with the same genes, for example troponin C skeletal muscle, IgM membrane heavy bound form, retinoic acid receptor gamma b and phosphofructokinase muscle b. Furthermore, primers were designed for 45 contigs annotated as tissue specific differentially expressed genes. For red muscle, these were follistatin-related protein 1, myoblast determination protein 2, nuclear receptor coactivator 4, growth hormone 2 and fatty acid binding protein 6. For white muscle, these were titin-like, four and a half LIM domains protein 1, ubiquitin specific protease 14, and heat shock protein 30. Primers to the targeted genes were designed using the Genamics Expression software and given in Gene Ontology Analysis For each tissue, Blast2GO was used to create DAT files containing: 1) the best BLAST hits from the filtered BLASTn vs. salmonid SIGENAE database but with accession numbers changed from SIGENAE to their best BLAST hit protein id in order to be able to retrieve GeneOntology terms; 2) the best BLAST hits from the filtered BLASTx vs. the D. rerio refSeq database and 3) the best BLAST hits and non-annotated sequences from the BLASTx vs. refSeq Metazoa Proteins database excluding D. rerio. 
These files were used to perform GO analysis on the whole transcriptome of red and white muscle. Specifically, both tissues were compared on biological processes, molecular functions and cel
