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      Analysis of Litopenaeus vannamei Transcriptome Using the Next-Generation DNA Sequencing Technique


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          Pacific white shrimp ( Litopenaeus vannamei), the major species of farmed shrimps in the world, has been attracting extensive studies, which require more and more genome background knowledge. The now available transcriptome data of L. vannamei are insufficient for research requirements, and have not been adequately assembled and annotated.

          Methodology/Principal Findings

          This is the first study that used a next-generation high-throughput DNA sequencing technique, the Solexa/Illumina GA II method, to analyze the transcriptome from whole bodies of L. vannamei larvae. More than 2.4 Gb of raw data were generated, and 109,169 unigenes with a mean length of 396 bp were assembled using the SOAP denovo software. 73,505 unigenes (>200 bp) with good quality sequences were selected and subjected to annotation analysis, among which 37.80% can be matched in NCBI Nr database, 37.3% matched in Swissprot, and 44.1% matched in TrEMBL. Using BLAST and BLAST2Go softwares, 11,153 unigenes were classified into 25 Clusters of Orthologous Groups of proteins (COG) categories, 8171 unigenes were assigned into 51 Gene ontology (GO) functional groups, and 18,154 unigenes were divided into 220 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. To primarily verify part of the results of assembly and annotations, 12 assembled unigenes that are homologous to many embryo development-related genes were chosen and subjected to RT-PCR for electrophoresis and Sanger sequencing analyses, and to real-time PCR for expression profile analyses during embryo development.


          The L. vannamei transcriptome analyzed using the next-generation sequencing technique enriches the information of L. vannamei genes, which will facilitate our understanding of the genome background of crustaceans, and promote the studies on L. vannamei.

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          Most cited references38

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          TGICL is a pipeline for analysis of large Expressed Sequence Tags (EST) and mRNA databases in which the sequences are first clustered based on pairwise sequence similarity, and then assembled by individual clusters (optionally with quality values) to produce longer, more complete consensus sequences. The system can run on multi-CPU architectures including SMP and PVM.
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              Systematic comparison of microarray profiling, real-time PCR, and next-generation sequencing technologies for measuring differential microRNA expression.

              RNA abundance and DNA copy number are routinely measured in high-throughput using microarray and next-generation sequencing (NGS) technologies, and the attributes of different platforms have been extensively analyzed. Recently, the application of both microarrays and NGS has expanded to include microRNAs (miRNAs), but the relative performance of these methods has not been rigorously characterized. We analyzed three biological samples across six miRNA microarray platforms and compared their hybridization performance. We examined the utility of these platforms, as well as NGS, for the detection of differentially expressed miRNAs. We then validated the results for 89 miRNAs by real-time RT-PCR and challenged the use of this assay as a "gold standard." Finally, we implemented a novel method to evaluate false-positive and false-negative rates for all methods in the absence of a reference method.

                Author and article information

                Role: Editor
                PLoS One
                PLoS ONE
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                11 October 2012
                : 7
                : 10
                [1 ]MOE Key Laboratory of Aquatic Product Safety/State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, People’s Republic of China
                [2 ]School of Marine Sciences, Sun Yat-sen University, Guangzhou, People’s Republic of China
                [3 ]Division of Cell Biology and Biophysics, School of Biological Science, University of Missouri-Kansas City, Kansas City, United States of America
                Biodiversity Insitute of Ontario - University of Guelph, Canada
                Author notes

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

                Conceived and designed the experiments: JH XX LC YX. Performed the experiments: XX CL. Analyzed the data: XX CL SW YC. Contributed reagents/materials/analysis tools: XX CL SW YC LL HZ. Wrote the manuscript: XX JH.


                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.

                Page count
                Pages: 12
                This research was supported by National Natural Science Foundation of China under grant No. U1131002 ( http://www.nsfc.gov.cn/Portal0/default106.htm); National High Technology Research and Development Program of China (973 Program) 2012CB114401 ( http://www.973.gov.cn/Default_3.aspx); China Agriculture Research System (47); Special Fund for Agro-scientific Research in the Public Interest 201103034 ( http://english.agri.gov.cn/); Foundation from Science and Technology Bureau of Guangdong Province 2011A020102002 and 2009A020102002 ( http://www.gdstc.gov.cn/eng/mission.html); Foundation from Administration of Ocean and Fisheries of Guangdong Province A201101B02 ( http://www.gdofa.gov.cn/index.php/English/); and the Open Project of the State Key Laboratory of Biocontrol (SKLBC09K04) ( http://sklbc.sysu.edu.cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Research Article
                Computational Biology
                Genome Analysis Tools
                Sequence Analysis
                Genome Analysis Tools
                Genome Sequencing
                Marine Biology
                Marine Technology
                Earth Sciences
                Marine and Aquatic Sciences
                Marine Technology
                Science Policy
                Technology Development



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