Project description:The largest of the tuna species, Atlantic bluefin tuna, Thunnus thynnus (Linnaeus, 1758), inhabits the North Atlantic Ocean and the Mediterranean Sea and is considered to be an endangered species, largely through overfishing. Thus, the development of aquaculture practices independent of wild resources can provide an important contribution towards ensuring security and sustainability of this species in the longer-term. In order to provide a resource for ongoing studies, we have used 454 pyrosequencing technology to sequence a mixed-tissue normalized cDNA library, derived from adult individuals. Transcript sequences were used to develop a novel 15K Agilent oligo microarray for T. thynnus and comparative tissue gene expression profiles were inferred for gill, heart, liver, ovaries and testes.
Project description:MicroRNAs (miRNAs) are a new class of small RNAs of approximately 22 nucleotides in length that control eukaryotic gene expression by fine tuning mRNA translation. They regulate a wide variety of biological processes, namely developmental timing, cell differentiation, cell proliferation, immune response and infection. For this reason, their identification is essential to understand eukaryotic biology. Their small size, low abundance and high instability complicated early identification; however, cloning/Sanger sequencing and new generation genome sequencing approaches overcame most technical hurdles and are being used for rapid miRNA identification in many eukaryotes. We have applied 454 DNA pyrosequencing technology to miRNA discovery in zebrafish (Danio rerio). For this, a series of cDNA libraries were prepared from small non-coding RNAs isolated at different embryonic time points and from fully developed organs. Each cDNA library was tagged with specific sequences and was sequenced using the Roche FLX genome sequencer. This approach retrieved 90% of the 192 miRNAs previously identified by cloning/Sanger sequencing and bioinformatics and 25 novel miRNAs were predicted.
Project description:Comparison of the endogenous small RNA content of Arabidopsis flower bud tissue: wild type vs. mutants in polIV pathways Keywords: High throughput 454 small RNA sequencing. Size fractionated small RNA from total RNA extracts was ligated to adapters, purified again and reverse transcribed. After PCR amplification the sample was subjected to 454 high throughput pyrosequencing. Please see www.454.com for details of the sequencing technology.
Project description:In this work, 454 pyrosequencing was used to build up a 3’ cDNA fragment library from a normalized library constructed from pooled RNA samples extracted at different stages of A. quisqualis mycoparasitization process (recognition, early and late parasitization). An extensive catalogue of unique transcripts was compiled and used to develop a microarray for large-scale analysis of genes involved in this mycoparasitism. We examined the transcriptomic changes that occur during the first stage of mycoparasitism (conidial germination). Our results showed that 1,776 transcripts are regulated during germination in the presence of powdery mildew. A striking feature of the gene catalogue was the presence of a number of genes predicted to encode proteins involved in the production of, glucanases, chitinases and extracellular proteases. This suggests that A. quisqualis causes the degradation of powdery mildew macromolecular constituents to provide the carbon skeletons and energy for the synthesis of proteins and other components destined for the developing of the mycelium.
Project description:In this work, 454 pyrosequencing was used to build up a 3M-bM-^@M-^Y cDNA fragment library from a normalized library constructed from pooled RNA samples extracted at different stages of A. quisqualis mycoparasitization process (recognition, early and late parasitization). An extensive catalogue of unique transcripts was compiled and used to develop a microarray for large-scale analysis of genes involved in this mycoparasitism. We examined the transcriptomic changes that occur during the first stage of mycoparasitism (conidial germination). Our results showed that 1,776 transcripts are regulated during germination in the presence of powdery mildew. A striking feature of the gene catalogue was the presence of a number of genes predicted to encode proteins involved in the production of, glucanases, chitinases and extracellular proteases. This suggests that A. quisqualis causes the degradation of powdery mildew macromolecular constituents to provide the carbon skeletons and energy for the synthesis of proteins and other components destined for the developing of the mycelium. Microarray oligo probes were designed based on 454 sequencing of 3'-ends of transcripts of a sample constituted by pooling RNAs extracted at different stages of A. quisqualis mycoparasitization process (recognition, early and late parasitization)