Deciphering the Transcriptome of Litopenaeus vannamei Using Next-Generation Sequencing

• Main Authors: Kun-Lin Li, 李昆霖
• Other Authors: Chung-Yen Lin
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/97315910881195840954

碩士 === 國立臺灣大學 === 漁業科學研究所 === 101 === Whiteleg shrimp (Litopenaeus vannamei), also known as Pacific white shrimp, is one of the major aquaculture species in Taiwan and around world, while the information of molecular regulation and basic gene knowledge on whiteleg shrimp are poor. In this study, we aim to apply Next-Generation Sequencing (NGS) technology to get the most updated and well annotated transcriptome of the Litopenaeus vannamei. Starting from 168 million illumina read pairs with average length around 90 bps from RNA-Seq libraries of post-larvae whole body (PL), hematopoietic tissue (HPT), hemocyte (HC), and larvae (composed by several development stages), we perform Trinity (de novo assembler) to assemble 190 thousand contigs in average length equal to 893 bps. For decreasing of redundant contigs, the assembly were further merged by CD-HIT. Finally, 165,000 contigs around were representative as whole transcriptome of whiteleg shrimp in this study. Sixty-two thousand putative protein products were predicted with at least length >30 residues. Overall 38.5% of the protein products were annotated by nr (E value cutoff: 1E-5). Among these annotated contigs, eighty percent of the best matched sequences are from Phylum Arthropoda. Meanwhile, forty percent of putative proteins can map on KEGG database. A single dose of lipopolysaccharide (LPS) administration was applied for mimicking the immune response upon bacteria challenge in shrimps. RNA-Seq data from hematopoietic tissue and hemocytes were mapped to the shrimp transcriptome assembly to calculate the expression profiling. The differentially expressed genes (DEGs) were identified and further clustered to reveal the specific expression patterns. In this approach, three important immune genes, Astakine, Antimicrobial peptide, and Prophenoloxidase, are found with high expressions in hemocyte. The expression of Astakine was peaked at six hours after LPS administration, but the expression of Antimicrobial peptide and Prophenoloxidase were in opposite way. Genes like fushi tarazu-factor 1 and zinc metalloproteinase were also found in the same expression patterns by the clustering analysis, suggesting the functional cooperation with Astakine in LPS induced response. Next-Generation Sequencing (NGS) undergoes vigorous development in recent years. Compare to the traditional sequencing, NGS has lower cost and can fetch a large amounts of data with the high efficiency and benefit. In this study, we successfully applied this approach and build a pipeline to recover almost all of the known white shrimp transcripts in NCBI Unigene set and discovered more non-protein coding genes. By integration of all the assembly with annotations, the web database for transcriptome of Litopenaeus vannamei is built and it will be shared to research community worldwide with free access.