Advanced Genetic Technologies, KY

High-throughput DNA sequencing has rapidly become a key tool in molecular biology, and hundreds of eukaryotic genomes as well as thousands of prokaryotic genomes have been sequenced to date. But, interpretation of the genomes requires accurate elucidation of the gene structures, for which in silico techniques remain limited by the complexity and diversity of gene expression processes, namely, transcription initiation and termination, and transcript processing. Furthermore, sequencing of transcriptomes — mRNAs and other cellular RNAs derived from transcription of genomic DNA — presents some additional challenges compared to genome sequencing. The University of Kentucky Advanced Genetic Technologies Center (UK-AGTC) provides at-cost highthroughput DNA sequencing and other high-throughput DNA analyses. Recently, UK-AGTC acquired a Roche/454 Life Sciences GS FLX/Titanium pyrosequencer, capable of sequencing greater than one million DNA fragments, averaging greater than 300 bases in length in a single oneday run. This is a very well established platform for low-cost sequencing of complete genomes and deep sequencing of DNA from environmental samples (metagenomes). However, its use for transcriptome sequencing, though promising, is not as well established. We propose to test a variety of strategies for transcriptome sequencing, and apply those techniques to several plants, microbes and insects of importance to agriculture and the environment.