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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 references 43

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          Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.

           K Livak,  T Schmittgen (2001)
          The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data. Copyright 2001 Elsevier Science (USA).
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            Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.

            The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons, a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using this matrix. The resulting Position-Specific Iterated BLAST (PSI-BLAST) program runs at approximately the same speed per iteration as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST is used to uncover several new and interesting members of the BRCT superfamily.
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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

                Affiliations
                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.

                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
                3820681 PONE-D-13-26397 10.1371/journal.pone.0079516

                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.

                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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