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      Transcriptome Analysis of the Innate Immunity-Related Complement System in Spleen Tissue of Ctenopharyngodon idella Infected with Aeromonas hydrophila

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          Abstract

          The grass carp ( Ctenopharyngodon idella) is an important commercial farmed herbivorous fish species in China, but is susceptible to Aeromonas hydrophila infections. In the present study, we performed de novo RNA-Seq sequencing of spleen tissue from specimens of a disease-resistant family, which were given intra-peritoneal injections containing PBS with or without a dose of A. hydrophila. The fish were sampled from the control group at 0 h, and from the experimental group at 4, 8, 12, 24, 48 and 72 h. 122.18 million clean reads were obtained from the normalized cDNA libraries; these were assembled into 425,260 contigs and then 191,795 transcripts. Of those, 52,668 transcripts were annotated with the NCBI Nr database, and 41,347 of the annotated transcripts were assigned into 90 functional groups. 20,569 unigenes were classified into six main categories, including 38 secondary KEGG pathways. 2,992 unigenes were used in the analysis of differentially expressed genes (DEGs). 89 of the putative DEGs were related to the immune system and 41 of them were involved in the complement and coagulation cascades pathway. This study provides insights into the complement and complement-related pathways involved in innate immunity, through expression profile analysis of the genomic resources in C. idella. We conclude that complement and complement-related genes play important roles during defense against A. hydrophila infection. The immune response is activated at 4 h after the bacterial injections, indicating that the complement pathways are activated at the early stage of bacterial infection. The study has improved our understanding of the immune response mechanisms in C. idella to bacterial pathogens.

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          Most cited references 42

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          Mapping and quantifying mammalian transcriptomes by RNA-Seq.

          We have mapped and quantified mouse transcriptomes by deeply sequencing them and recording how frequently each gene is represented in the sequence sample (RNA-Seq). This provides a digital measure of the presence and prevalence of transcripts from known and previously unknown genes. We report reference measurements composed of 41-52 million mapped 25-base-pair reads for poly(A)-selected RNA from adult mouse brain, liver and skeletal muscle tissues. We used RNA standards to quantify transcript prevalence and to test the linear range of transcript detection, which spanned five orders of magnitude. Although >90% of uniquely mapped reads fell within known exons, the remaining data suggest new and revised gene models, including changed or additional promoters, exons and 3' untranscribed regions, as well as new candidate microRNA precursors. RNA splice events, which are not readily measured by standard gene expression microarray or serial analysis of gene expression methods, were detected directly by mapping splice-crossing sequence reads. We observed 1.45 x 10(5) distinct splices, and alternative splices were prominent, with 3,500 different genes expressing one or more alternate internal splices.
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            Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research.

            We present here Blast2GO (B2G), a research tool designed with the main purpose of enabling Gene Ontology (GO) based data mining on sequence data for which no GO annotation is yet available. B2G joints in one application GO annotation based on similarity searches with statistical analysis and highlighted visualization on directed acyclic graphs. This tool offers a suitable platform for functional genomics research in non-model species. B2G is an intuitive and interactive desktop application that allows monitoring and comprehension of the whole annotation and analysis process. Blast2GO is freely available via Java Web Start at http://www.blast2go.de. http://www.blast2go.de -> Evaluation.
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              Velvet: algorithms for de novo short read assembly using de Bruijn graphs.

              We have developed a new set of algorithms, collectively called "Velvet," to manipulate de Bruijn graphs for genomic sequence assembly. A de Bruijn graph is a compact representation based on short words (k-mers) that is ideal for high coverage, very short read (25-50 bp) data sets. Applying Velvet to very short reads and paired-ends information only, one can produce contigs of significant length, up to 50-kb N50 length in simulations of prokaryotic data and 3-kb N50 on simulated mammalian BACs. When applied to real Solexa data sets without read pairs, Velvet generated contigs of approximately 8 kb in a prokaryote and 2 kb in a mammalian BAC, in close agreement with our simulated results without read-pair information. Velvet represents a new approach to assembly that can leverage very short reads in combination with read pairs to produce useful assemblies.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                6 July 2016
                2016
                : 11
                : 7
                Affiliations
                [1 ]Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University, Ministry of Education, Shanghai 201306, PR China
                [2 ]National Pathogen Collection Center for Aquatic Animals, College of Fisheries and Life Science, Shanghai Ocean University, 999 Huchenghuan Road, Shanghai 201306, PR China
                Chinese Academy of Fishery Sciences, CHINA
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: YFD JLL LQL. Performed the experiments: YFD XYX. Analyzed the data: YFD. Contributed reagents/materials/analysis tools: YFD YBS MYH MZ LSL. Wrote the paper: YFD JLL.

                Article
                PONE-D-16-13569
                10.1371/journal.pone.0157413
                4934786
                27383749
                © 2016 Dang et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Counts
                Figures: 9, Tables: 5, Pages: 22
                Product
                Funding
                Funded by: The China's Agricultural Reseach System
                Award ID: CARS-46-04
                Award Recipient :
                Funded by: The National Key Technology R and D Program of China
                Award ID: 2012BAD26B02
                Award Recipient :
                The work was supported by the following: the China's Agricultural Research System (CARS-46-04), Jiale Li; and the National Key Technology R&D Program of China (2012BAD26B02), Jiale Li.
                Categories
                Research Article
                Biology and Life Sciences
                Physiology
                Immune Physiology
                Complement System
                Medicine and Health Sciences
                Physiology
                Immune Physiology
                Complement System
                Biology and Life Sciences
                Immunology
                Immune System
                Complement System
                Medicine and Health Sciences
                Immunology
                Immune System
                Complement System
                Biology and Life Sciences
                Immunology
                Immune System Proteins
                Complement System
                Medicine and Health Sciences
                Immunology
                Immune System Proteins
                Complement System
                Biology and Life Sciences
                Biochemistry
                Proteins
                Immune System Proteins
                Complement System
                Biology and Life Sciences
                Immunology
                Immune System
                Medicine and Health Sciences
                Immunology
                Immune System
                Biology and Life Sciences
                Organisms
                Bacteria
                Aeromonas
                Aeromonas Hydrophila
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
                Bacterial Pathogens
                Aeromonas Hydrophila
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Microbial Pathogens
                Bacterial Pathogens
                Aeromonas Hydrophila
                Biology and Life Sciences
                Organisms
                Animals
                Vertebrates
                Fishes
                Osteichthyes
                Carps
                Biology and Life Sciences
                Genetics
                Gene Expression
                Biology and Life Sciences
                Organisms
                Plants
                Grasses
                Research and Analysis Methods
                Database and Informatics Methods
                Biological Databases
                Sequence Databases
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Sequence Databases
                Research and Analysis Methods
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Sequence Databases
                Research and analysis methods
                Extraction techniques
                RNA extraction
                Custom metadata
                All transcriptome data files are available from the NCBI Sequence Read Archive database (accession number SRP060308).

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