Functional annotations of the unigenes of <i>C. idella</i>.

<p>(A) KOG annotation. (B) Level 2 GO term distribution for the biological process, cellular component and molecular function categories.</p

Statistics of the annotation results for the <i>C. idella</i> unigenes.

<p>Nr: NCBI non-redundant protein sequences, Nt: NCBI non-redundant nucleotide sequences, Pfam: Protein family, KOG: Clusters of Orthologous Groups of proteins, Swiss-Prot: A manually annotated and reviewed protein sequence database, KO: KEGG Ortholog database and GO: Gene Ontology.</p><p>Statistics of the annotation results for the <i>C. idella</i> unigenes.</p

<i>De novo</i> Assembly of the Grass Carp <i>Ctenopharyngodon idella</i> Transcriptome to Identify miRNA Targets Associated with Motile Aeromonad Septicemia

<div><p>Background</p><p><i>De novo</i> transcriptome sequencing is a robust method of predicting miRNA target genes, especially for organisms without reference genomes. Differentially expressed miRNAs had been identified previously in kidney samples collected from susceptible and resistant grass carp (<i>Ctenopharyngodon idella</i>) affected by <i>Aeromonas hydrophila</i>. Target identification for these differentially expressed miRNAs poses a major challenge in this non-model organism.</p><p>Results</p><p>Two cDNA libraries constructed from mRNAs of susceptible and resistant <i>C. idella</i> were sequenced by Illumina Hiseq 2000 technology. A total of more than 100 million reads were generated and <i>de novo</i> assembled into 199,593 transcripts which were further extensively annotated by comparing their sequences to different protein databases. Biochemical pathways were predicted from these transcript sequences. A BLASTx analysis against a non-redundant protein database revealed that 61,373 unigenes coded for 28,311 annotated proteins. Two cDNA libraries from susceptible and resistant samples showed that 721 unigenes were expressed at significantly different levels; 475 were significantly up-regulated and 246 were significantly down-regulated in the SG samples compared to the RG samples. The computational prediction of miRNA targets from these differentially expressed genes identified 188 unigenes as the targets of 5 conserved and 4 putative novel miRNA families.</p><p>Conclusion</p><p>This study demonstrates the feasibility of identifying miRNA targets by transcriptome analysis. The transcriptome assembly data represent a substantial increase in the genomic resources available for <i>C. idella</i> and will provide insights into the gene expression profile analysis and the miRNA function annotations in further studies.</p></div

Summaries of sequencing cDNA library.

<p>Summaries of sequencing cDNA library.</p

Differentially expressed miRNAs in <i>C. idella</i> kidney between SG and RG.

<p>Differentially expressed miRNAs in <i>C. idella</i> kidney between SG and RG.</p

Top 10 list of pathways related to immune system.

<p>Top 10 list of pathways related to immune system.</p

Top 10 list of the gene number of Pathway Hierarchy 2.

<p>Top 10 list of the gene number of Pathway Hierarchy 2.</p

26 pathways were related to immune and diseases in all pathways.

<p><b>List of gene abbreviations</b>: JNK: c-Jun N-terminal kinase, TLR5: toll-like receptor 5, TBK1: TANK-binding kinase 1, LDLR: low-density lipoprotein receptor, FYN: tyrosine-protein kinase Fyn, CTSS: cathepsin S, PLK3: polo-like kinase 3, DNAJB1: DnaJ homolog subfamily B member 1, MKP: dual specificity MAP kinase phosphatase, DOCK2: dedicator of cytokinesis protein 2, ADCY7: adenylate cyclase 7, SLC2A1: MFS transporter, SP family, solute carrier family 2 (facilitated glucose transporter), member 1, CCR7: C-C chemokine receptor type 7, TNFSF12: tumor necrosis factor ligand superfamily member 12, CFB: component factor B.</p><p>26 pathways were related to immune and diseases in all pathways.</p

Number and Fold change distribution of differentially expressed genes between the SG/RG libraries.

<p>Number and Fold change distribution of differentially expressed genes between the SG/RG libraries.</p

The expression analysis of selected genes from the expression profile by relative quantitative real-time PCR.

<p>A Transcriptome sequencing data, B Real-time PCR data. Increases and decreases in relative levels of transcripts with respect to the control 18s for mRNA and <i>miR-22a</i> for miRNA are shown. The settings “q.value <0.005” and “|log2.Fold change.normalized|>2” were used as thresholds for judging significant differences in transcript expression. One-way ANOVA tests were performed using SPSS 17.0 to determine significant differences for Real-time PCR data. Statistical significance of the relative expression ratio is indicated *.</p