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Advanced Genetic Technologies, KY
Department of Plant Pathology
The genome of an organism represents the total of all genes, which are encoded in the base sequences of DNA of that organism. In order for a gene to be expressed, it must first be transcribed into RNA molecules, each similar in sequence to a DNA segment. Then, the RNAs are spliced and processed into mRNAs, which are then translated to give functional proteins. The entire set of mRNAs in a cell, tissue or organism is called its transcriptome. Analysis of whole-transcriptome sequence is key to understanding how genomes direct the organism's form, development, behavior, and responses to the environment. New technologies such as pyrosequencing have greatly increased DNA sequencing rates and reduced costs. In this study, several strategies will be tested for pyrosequencing of transcriptomes. Once these protocols are refined, they will be applied to various organisms of relevance to agricultural research, including plants, insects, and various pathogens and parasites. The results will serve as preliminary data for competitively funded research programs.
2011 Project Description
Several RNA (transcriptome) sequencing projects were undertaken on plants, insects, fungi, and symbiotic microbes responsible for cellulose digestion in wood roaches and termites. Two methods were compared: the Roche/454 rapid cDNA sequencing system, and the Matz protocol (Meyer E, Aglyamova G, Wang S, Buchanan-Carter J, Abrego D, Colbourne J, Willis B, Matz M (2009) Sequencing and de novo analysis of a coral larval transcriptome using 454 GSFlx. BMC Genomics 10: 219.)
Also, assembly programs for transcriptome sequence were compared, including Velvet, CLC Genome Workbench, and MapSplice.
Finally, the bioinformatition assigned to the AGTC received training in the Blast2GO genome and transcriptome annotation system.
The Roche/454 rapid cDNA sequencing system proved to be feasible for most applications. The Matz protocol was considerably more expensive and time-consuming, but provided more specific information that enhanced accuracy of gene models generated from the data. Strengths and weaknesses of the various software packages were also identified. The results of these studies provide a basis for standard operating procedures and recommended protocols to be used by the AGTC core facility
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