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      De novo assembly of the Carcinus maenas transcriptome and characterization of innate immune system pathways

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          Abstract

          Background

          The European shore crab, Carcinus maenas, is used widely in biomonitoring, ecotoxicology and for studies into host-pathogen interactions. It is also an important invasive species in numerous global locations. However, the genomic resources for this organism are still sparse, limiting research progress in these fields. To address this resource shortfall we produced a C. maenas transcriptome, enabled by the progress in next-generation sequencing technologies, and applied this to assemble information on the innate immune system in this species.

          Results

          We isolated and pooled RNA for twelve different tissues and organs from C. maenas individuals and sequenced the RNA using next generation sequencing on an Illumina HiSeq 2500 platform. After de novo assembly a transcriptome was generated encompassing 212,427 transcripts (153,699 loci). The transcripts were filtered, annotated and characterised using a variety of tools (including BLAST, MEGAN and RSEM) and databases (including NCBI, Gene Ontology and KEGG). There were differential patterns of expression for between 1,223 and 2,741 transcripts across tissues and organs with over-represented Gene Ontology terms relating to their specific function. Based on sequence homology to immune system components in other organisms, we show both the presence of transcripts for a series of known pathogen recognition receptors and response proteins that form part of the innate immune system, and transcripts representing the RNAi, Toll-like receptor signalling, IMD and JAK/STAT pathways.

          Conclusions

          We have produced an assembled transcriptome for C. maenas that provides a significant molecular resource for wide ranging studies in this species. Analysis of the transcriptome has revealed the presence of a series of known targets and functional pathways that form part of their innate immune system and illustrate tissue specific differences in their expression patterns.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s12864-015-1667-1) contains supplementary material, which is available to authorized users.

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          Most cited references41

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          Virus entry by endocytosis.

          Although viruses are simple in structure and composition, their interactions with host cells are complex. Merely to gain entry, animal viruses make use of a repertoire of cellular processes that involve hundreds of cellular proteins. Although some viruses have the capacity to penetrate into the cytosol directly through the plasma membrane, most depend on endocytic uptake, vesicular transport through the cytoplasm, and delivery to endosomes and other intracellular organelles. The internalization may involve clathrin-mediated endocytosis (CME), macropinocytosis, caveolar/lipid raft-mediated endocytosis, or a variety of other still poorly characterized mechanisms. This review focuses on the cell biology of virus entry and the different strategies and endocytic mechanisms used by animal viruses.
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            Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways.

            The RNase III enzyme Dicer processes RNA into siRNAs and miRNAs, which direct a RNA-induced silencing complex (RISC) to cleave mRNA or block its translation (RNAi). We have characterized mutations in the Drosophila dicer-1 and dicer-2 genes. Mutation in dicer-1 blocks processing of miRNA precursors, whereas dicer-2 mutants are defective for processing siRNA precursors. It has been recently found that Drosophila Dicer-1 and Dicer-2 are also components of siRNA-dependent RISC (siRISC). We find that Dicer-1 and Dicer-2 are required for siRNA-directed mRNA cleavage, though the RNase III activity of Dicer-2 is not required. Dicer-1 and Dicer-2 facilitate distinct steps in the assembly of siRISC. However, Dicer-1 but not Dicer-2 is essential for miRISC-directed translation repression. Thus, siRISCs and miRISCs are different with respect to Dicers in Drosophila.
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              Biases in Illumina transcriptome sequencing caused by random hexamer priming

              Generation of cDNA using random hexamer priming induces biases in the nucleotide composition at the beginning of transcriptome sequencing reads from the Illumina Genome Analyzer. The bias is independent of organism and laboratory and impacts the uniformity of the reads along the transcriptome. We provide a read count reweighting scheme, based on the nucleotide frequencies of the reads, that mitigates the impact of the bias.
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                Author and article information

                Contributors
                bv213@exeter.ac.uk
                L.K.Bickley@exeter.ac.uk
                e.santos@exeter.ac.uk
                c.r.tyler@exeter.ac.uk
                grant.stentiford@cefas.co.uk
                kelly.bateman@cefas.co.uk
                ronny.vanaerle@cefas.co.uk
                Journal
                BMC Genomics
                BMC Genomics
                BMC Genomics
                BioMed Central (London )
                1471-2164
                16 June 2015
                16 June 2015
                2015
                : 16
                : 1
                : 458
                Affiliations
                [ ]Biosciences, College of Life & Environmental Sciences, University of Exeter, Geoffrey Pope Building, Exeter, EX4 4QD UK
                [ ]European Union Reference Laboratory for Crustacean Diseases, Centre for Environment, Fisheries and Aquaculture Science (Cefas), Weymouth Laboratory, Weymouth, Dorset DT4 8UB UK
                [ ]Aquatic Health and Hygiene Division, Centre for Environment, Fisheries and Aquaculture Science (Cefas), Weymouth Laboratory, Weymouth, Dorset DT4 8UB UK
                Article
                1667
                10.1186/s12864-015-1667-1
                4469326
                26076827
                0ebfd3b3-5ac6-4ef2-939e-ed6d4dd70e18
                © Verbruggen et al. 2015

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 2 December 2014
                : 29 May 2015
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2015

                Genetics
                Genetics

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