The libraries were loaded onto flow cell channels for sequencing on the Illumina HiSeq 2000 in strument by the Chinese National Human Genome Cen ter. A total Dovitinib structure of six paired end cDNA libraries of zebrafish livers were constructed for each of the test groups of WED immunized and mock immunized fish. Triplicate biological replicates were per formed for each group. Raw data were deposited in the NCBI database under submission num ber SRA048658. 2. Transcriptome analysis The Illumina HiSeq 2000 system generated 120 bp raw PE reads were first processed by the FASTX Toolkit to remove the reads with sequencing adaptors and of low quality. Then, the Burrows Wheeler Aligners Smith Waterman Alignment Inhibitors,Modulators,Libraries program was used to align the remaining reads to the reference zebrafish mRNA from the Ensembl database.
The transcription level of each gene was deduced by deter mining the total number of reads mapped to each gene using Picard tools. Dif ferentially expressed genes were identified by the DESeq package in R software, using two fold change 1 or 1 and p value 0. 05 as the threshold. After data normalization by the p Inhibitors,Modulators,Libraries value and FDR calculation, the resulting expression intensity values were analyzed by the MA plot based method, as described by Wang et al. Functional analysis of differentially expressed genes The Database for Annotation, Visualization and Inte grated Discovery was used to investi gate functional enrichment Inhibitors,Modulators,Libraries for over and under expressed genes by more than two fold in the WED immunized group relative to the mock immunized group.
Gene functional enrichment was performed using the default parameters Inhibitors,Modulators,Libraries in DAVID to obtain an adjusted p value 0. 05 for the test gene group versus the zebra fish gene ontology annotation set. The fold enrichment cut off suggested for DAVID functional annotation is 1. 5. In addition, the significantly up regulated genes from the differentially expressed genes dataset were further analyzed by Inhibitors,Modulators,Libraries investigating the corresponding GO biological processes. Furthermore, GO analysis of genes transcribed at different levels was also performed using the Biological Networks Gene Ontology tool, which is based on the Cytoscape software. The hypergeometric test with Benjamini Hochberg False Discovery Rate was performed using the default parameters to obtain an adjusted p value between the test gene group and the merged non redundancy zebrafish and mouse GO annotation set. Finally, the web based Kyoto Encyclopedia of Genes and Gen omes pathway analysis program run by the KEGG certainly Automatic Annotation Server was used to obtain func tional annotation of genes by performing basic local alignment search tool mediated comparisons against the manually curated KEGG GENES database.