Characterization of the rainbow trout transcriptome using Sanger and 454-pyrosequencing approaches (original) (raw)
Related papers
2012
Rainbow trout, Oncorhynchus mykiss, is an important aquaculture species worldwide and, in addition to being of commercial interest, it is also a research model organism of considerable scientific importance. Because of the lack of a whole genome sequence in that species, transcriptomic analyses of this species have often been hindered. Using next-generation sequencing (NGS) technologies, we sought to fill these informational gaps. Here, using Roche 454-Titanium technology, we provide new tissue-specific cDNA repertoires from several rainbow trout tissues. Non-normalized cDNA libraries were constructed from testis, ovary, brain and gill rainbow trout tissue samples, and these different libraries were sequenced in 10 separate half-runs of 454-Titanium. Overall, we produced a total of 3 million quality sequences with an average size of 328 bp, representing more than 1 Gb of expressed sequence information. These sequences have been combined with all publicly available rainbow trout sequences, resulting in a total of 242,187 clusters of putative transcript groups and 22,373 singletons. To identify the predominantly expressed genes in different tissues of interest, we developed a Digital Differential Display (DDD) approach. This approach allowed us to characterize the genes that are predominantly expressed within each tissue of interest. Of these genes, some were already known to be tissue-specific, thereby validating our approach. Many others, however, were novel candidates, demonstrating the usefulness of our strategy and of such tissue-specific resources. This new sequence information, acquired using NGS 454-Titanium technology, deeply enriched our current knowledge of the expressed genes in rainbow trout through the identification of an increased number of tissue-specific sequences. This identification allowed a precise cDNA tissue repertoire to be characterized in several important rainbow trout tissues. The rainbow trout contig browser can be accessed at the following publicly available web site (http://www.sigenae.org/).
Status and opportunities for genomics research with rainbow trout
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2002
The rainbow trout (Oncorhynchus mykiss) is one of the most widely studied of model fish species. Extensive basic biological information has been collected for this species, which because of their large size relative to other model fish species are particularly suitable for studies requiring ample quantities of specific cells and tissue types. Rainbow trout have been widely utilized for research in carcinogenesis, toxicology, comparative immunology, disease ecology, physiology and nutrition. They are distinctive in having evolved from a relatively recent tetraploid event, resulting in a high incidence of duplicated genes. Natural populations are available and have been well characterized for chromosomal, protein, molecular and quantitative genetic variation. Their ease of culture, and experimental and aquacultural significance ૾ Contribution to a special issue of CBP on Comparative Functional Genomics. (G.H. Thorgaard). Numerous microsatellites have been isolated and two relatively detailed genetic maps have been developed. Extensive sequencing of expressed sequence tags has begun and four BAC libraries have been developed. The development and analysis of additional genomic sequence data will provide distinctive opportunities to address problems in areas such as evolution of the immune system and duplicate genes. ᮊ
A first generation integrated map of the rainbow trout genome
BMC Genomics, 2011
Background Rainbow trout (Oncorhynchus mykiss) are the most-widely cultivated cold freshwater fish in the world and an important model species for many research areas. Coupling great interest in this species as a research model with the need for genetic improvement of aquaculture production efficiency traits justifies the continued development of genomics research resources. Many quantitative trait loci (QTL) have been identified for production and life-history traits in rainbow trout. An integrated physical and genetic map is needed to facilitate fine mapping of QTL and the selection of positional candidate genes for incorporation in marker-assisted selection (MAS) programs for improving rainbow trout aquaculture production. Results The first generation integrated map of the rainbow trout genome is composed of 238 BAC contigs anchored to chromosomes of the genetic map. It covers more than 10% of the genome across segments from all 29 chromosomes. Anchoring of 203 contigs to chromosomes of the National Center for Cool and Cold Water Aquaculture (NCCCWA) genetic map was achieved through mapping of 288 genetic markers derived from BAC end sequences (BES), screening of the BAC library with previously mapped markers and matching of SNPs with BES reads. In addition, 35 contigs were anchored to linkage groups of the INRA (French National Institute of Agricultural Research) genetic map through markers that were not informative for linkage analysis in the NCCCWA mapping panel. The ratio of physical to genetic linkage distances varied substantially among chromosomes and BAC contigs with an average of 3,033 Kb/cM. Conclusions The integrated map described here provides a framework for a robust composite genome map for rainbow trout. This resource is needed for genomic analyses in this research model and economically important species and will facilitate comparative genome mapping with other salmonids and with model fish species. This resource will also facilitate efforts to assemble a whole-genome reference sequence for rainbow trout.
Genes
Selective breeding can significantly improve the establishment of sustainable and profitable aquaculture fish farming. For rainbow trout (Oncorhynchus mykiss), one of the main aquaculture coldwater species in Europe, a variety of selected hatchery strains are commercially available. In this study, we investigated the genetic variation between the local Born strain, selected for survival, and the commercially available Silver Steelhead strain, selected for growth. We sequenced the transcriptome of six tissues (gills, head kidney, heart, liver, spleen, and white muscle) from eight healthy individuals per strain, using RNA-seq technology to identify strain-specific gene-expression patterns and single nucleotide polymorphisms (SNPs). In total, 1760 annotated genes were differentially expressed across all tissues. Pathway analysis assigned them to different gene networks. We also identified a set of SNPs, which are heterozygous for one of the two breeding strains: 1229 of which represent...
Journal of Fish Biology, 2008
A rainbow trout high-density oligonucleotide microarray was constructed using all tentative consensus (TC) sequences that are publicly available from all international rainbow trout Oncorhynchus mykiss genomic research projects through the Rainbow Trout Gene Index database. The new array contains 60-mer oligonucleotide probes representing 37 394 unique TC sequences and 1417 control spots. The array (4 Â 44 format) was manufactured according to the design by Agilent Technologies using the inkjet-based SurePrint technology (design number 016320). The performance of the new microarray platform was evaluated by analysing gene expression associated with rainbow trout, vitellogenesis-induced muscle atrophy. This microarray will open new avenues of research that will aid in the development of novel strategies for genetic improvement for economically important traits benefiting the salmonid aquaculture industries.
Advocating Complete Sequencing of the Genome of the Rainbow Trout , Oncorhynchus mykiss
2002
Gary Thorgaard a *, George Bailey , David Williams , Donald Buhler , Stephen Kaattari , Sandra Ristow , John Hansen , James Winton , Jerri Bartholomew , James Nagler , Patrick Walsh , Matt Vijayan , Robert Devlin , Ronald Hardy , Kenneth Overturf , William Young , Barrie Robison , Caird Rexroad III , Yniv Palti p , Bernie May, Scott LaPatra, Ruth Phillips, Linda Park, Takashi Sakamoto, Nobuaki Okamoto, Roy Danzmann, Fred Allendorf, Lars-Erik Holm, Robert Bogden , Patricia Iturra, Rene Guyomard, Yann Guiguen (addresses of authors provided at end of paper) Corresponding author. Tel.: 509-335-7438; Fax: 509-335-3184; Email : thorglab @ wsu.edu
BMC Research Notes
Objectives The Brown trout is a salmonid species with a high commercial value in Europe. Life history and spawning behaviour include resident (Salmo trutta m. fario) and migratory (Salmo trutta m. trutta) ecotypes. The main objective is to apply RNA-seq technology in order to obtain a reference transcriptome of two key tissues, brain and muscle, of the riverine trout Salmo trutta m. fario. Having a reference transcriptome of the resident form will complement genomic resources of salmonid species. Data description We generate two cDNA libraries from pooled RNA samples, isolated from muscle and brain tissues of adult individuals of Salmo trutta m. fario, which were sequenced by Illumina technology. Raw reads were subjected to de-novo transcriptome assembly using Trinity, and coding regions were predicted by TransDecoder. A final set of 35,049 non-redundant ORF unigenes were annotated. Tissue differential expression analysis was evaluated by Cuffdiff. A False Discovery Rate (FDR) ≤ 0.0...
BMC genomics, 2006
Within the framework of a genomics project on livestock species (AGENAE), we initiated a high-throughput DNA sequencing program of Expressed Sequence Tags (ESTs) in rainbow trout, Oncorhynchus mykiss. We constructed three cDNA libraries including one highly complex pooled-tissue library. These libraries were normalized and subtracted to reduce clone redundancy. ESTs sequences were produced, and 96,472 ESTs corresponding to high quality sequence reads were released on the international database, currently representing 42.5% of the overall sequence knowledge in this species. All these EST sequences and other publicly available ESTs in rainbow trout have been included on a publicly available Website (SIGENAE) and have been clustered into a total of 52,930 clusters of putative transcripts groups, including 24,616 singletons. 57.1% of these 52,930 clusters are represented by at least one Agenae EST and 14,343 clusters (27.1%) are only composed by Agenae ESTs. Sequence analysis also revea...
BMC genomics, 2017
We describe an emerging initiative - the 'Functional Annotation of All Salmonid Genomes' (FAASG), which will leverage the extensive trait diversity that has evolved since a whole genome duplication event in the salmonid ancestor, to develop an integrative understanding of the functional genomic basis of phenotypic variation. The outcomes of FAASG will have diverse applications, ranging from improved understanding of genome evolution, to improving the efficiency and sustainability of aquaculture production, supporting the future of fundamental and applied research in an iconic fish lineage of major societal importance.
BMC Genomics, 2009
Background To enhance capabilities for genomic analyses in rainbow trout, such as genomic selection, a large suite of polymorphic markers that are amenable to high-throughput genotyping protocols must be identified. Expressed Sequence Tags (ESTs) have been used for single nucleotide polymorphism (SNP) discovery in salmonids. In those strategies, the salmonid semi-tetraploid genomes often led to assemblies of paralogous sequences and therefore resulted in a high rate of false positive SNP identification. Sequencing genomic DNA using primers identified from ESTs proved to be an effective but time consuming methodology of SNP identification in rainbow trout, therefore not suitable for high throughput SNP discovery. In this study, we employed a high-throughput strategy that used pyrosequencing technology to generate data from a reduced representation library constructed with genomic DNA pooled from 96 unrelated rainbow trout that represent the National Center for Cool and Cold Water Aquaculture (NCCCWA) broodstock population. Results The reduced representation library consisted of 440 bp fragments resulting from complete digestion with the restriction enzyme HaeIII; sequencing produced 2,000,000 reads providing an average 6 fold coverage of the estimated 150,000 unique genomic restriction fragments (300,000 fragment ends). Three independent data analyses identified 22,022 to 47,128 putative SNPs on 13,140 to 24,627 independent contigs. A set of 384 putative SNPs, randomly selected from the sets produced by the three analyses were genotyped on individual fish to determine the validation rate of putative SNPs among analyses, distinguish apparent SNPs that actually represent paralogous loci in the tetraploid genome, examine Mendelian segregation, and place the validated SNPs on the rainbow trout linkage map. Approximately 48% (183) of the putative SNPs were validated; 167 markers were successfully incorporated into the rainbow trout linkage map. In addition, 2% of the sequences from the validated markers were associated with rainbow trout transcripts. Conclusion The use of reduced representation libraries and pyrosequencing technology proved to be an effective strategy for the discovery of a high number of putative SNPs in rainbow trout; however, modifications to the technique to decrease the false discovery rate resulting from the evolutionary recent genome duplication would be desirable.