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      Sequencing and De Novo Assembly of the Asian Clam ( Corbicula fluminea) Transcriptome Using the Illumina GAIIx Method

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          Abstract

          Background

          The Asian clam ( Corbicula fluminea) is currently one of the most economically important aquatic species in China and has been used as a test organism in many environmental studies. However, the lack of genomic resources, such as sequenced genome, expressed sequence tags (ESTs) and transcriptome sequences has hindered the research on C. fluminea. Recent advances in large-scale RNA-Seq enable generation of genomic resources in a short time, and provide large expression datasets for functional genomic analysis.

          Methodology/Principal Findings

          We used a next-generation high-throughput DNA sequencing technique with an Illumina GAIIx method to analyze the transcriptome from the whole bodies of C. fluminea. More than 62,250,336 high-quality reads were generated based on the raw data, and 134,684 unigenes with a mean length of 791 bp were assembled using the Velvet and Oases software. All of the assembly unigenes were annotated by running BLASTx and BLASTn similarity searches on the Nt, Nr, Swiss-Prot, COG and KEGG databases. In addition, the Clusters of Orthologous Groups (COGs), Gene Ontology (GO) terms and Kyoto Encyclopedia of Gene and Genome (KEGG) annotations were also assigned to each unigene transcript. To provide a preliminary verification of the assembly and annotation results, and search for potential environmental pollution biomarkers, 15 functional genes (five antioxidase genes, two cytochrome P450 genes, three GABA receptor-related genes and five heat shock protein genes) were cloned and identified. Expressions of the 15 selected genes following fluoxetine exposure confirmed that the genes are indeed linked to environmental stress.

          Conclusions/Significance

          The C. fluminea transcriptome advances the underlying molecular understanding of this freshwater clam, provides a basis for further exploration of C. fluminea as an environmental test organism and promotes further studies on other bivalve organisms.

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          Most cited references33

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          Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology.

          Molecular chaperones, including the heat-shock proteins (Hsps), are a ubiquitous feature of cells in which these proteins cope with stress-induced denaturation of other proteins. Hsps have received the most attention in model organisms undergoing experimental stress in the laboratory, and the function of Hsps at the molecular and cellular level is becoming well understood in this context. A complementary focus is now emerging on the Hsps of both model and nonmodel organisms undergoing stress in nature, on the roles of Hsps in the stress physiology of whole multicellular eukaryotes and the tissues and organs they comprise, and on the ecological and evolutionary correlates of variation in Hsps and the genes that encode them. This focus discloses that (a) expression of Hsps can occur in nature, (b) all species have hsp genes but they vary in the patterns of their expression, (c) Hsp expression can be correlated with resistance to stress, and (d) species' thresholds for Hsp expression are correlated with levels of stress that they naturally undergo. These conclusions are now well established and may require little additional confirmation; many significant questions remain unanswered concerning both the mechanisms of Hsp-mediated stress tolerance at the organismal level and the evolutionary mechanisms that have diversified the hsp genes.
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            Next-generation transcriptome assembly.

            Transcriptomics studies often rely on partial reference transcriptomes that fail to capture the full catalogue of transcripts and their variations. Recent advances in sequencing technologies and assembly algorithms have facilitated the reconstruction of the entire transcriptome by deep RNA sequencing (RNA-seq), even without a reference genome. However, transcriptome assembly from billions of RNA-seq reads, which are often very short, poses a significant informatics challenge. This Review summarizes the recent developments in transcriptome assembly approaches - reference-based, de novo and combined strategies - along with some perspectives on transcriptome assembly in the near future.
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              Transcriptomics in the RNA-seq era.

              The transcriptomics field has developed rapidly with the advent of next-generation sequencing technologies. RNA-seq has now displaced microarrays as the preferred method for gene expression profiling. The comprehensive nature of the data generated has been a boon in terms of transcript identification but analysis challenges remain. Key among these problems is the development of suitable expression metrics for expression level comparisons and methods for identification of differentially expressed genes (and exons). Several approaches have been developed but as yet no consensus exists on the best pipeline to use. De novo transcriptome approaches are increasingly viable for organisms lacking a sequenced genome. The reduction in starting RNA required has enabled the development of new applications such as single cell transcriptomics. The emerging picture of mammalian transcription is complex with further refinement expected with the integration of epigenomic data generated by projects such as ENCODE. Copyright © 2013 Elsevier Ltd. All rights reserved.
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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, USA )
                1932-6203
                2013
                7 November 2013
                : 8
                : 11
                : e79516
                Affiliations
                [1]State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China
                National University of Singapore, Singapore
                Author notes

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

                Conceived and designed the experiments: HC JZ ZW. Performed the experiments: HC XL JB MW. Analyzed the data: HC XL. Contributed reagents/materials/analysis tools: HC JB. Wrote the manuscript: HC ZW.

                Article
                PONE-D-13-26397
                10.1371/journal.pone.0079516
                3820681
                24244519
                6ce4f8ec-d889-40d8-8417-fd6405d2aedf
                Copyright @ 2013

                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.

                History
                : 25 June 2013
                : 28 September 2013
                Funding
                This work was supported by National Basic Research Program of China (2009CB421605); the National Natural Science Foundation of China (21007086); the National High-tech R&D Program (2012AA06A302). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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