Plant, Animal & Microbe Genomes X Conference

January 12-16, 2002
Town & Country Convention Center
San Diego, CA

Poster: Genome Sequencing & ESTs
            

THE UK'S PUBLIC WHEAT TRANSCRIPTOME PROJECT - DEVELOPMENT OF A HIGH THROUGHPUT EST SEQUENCING PROTOCOL

¹ IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, BS41 9AF, UK.

² School of Biological Sciences, University of Bristol, Woodland Road, Bristol, BS8 1UG, UK.

As part of the BBSRC Investigating Gene Function (IGF) cereal program we aim to generate a winter wheat (var. Mercia), EST, unigene resource for the production of high density microarray chips. To create these the cDNAs of 1000 randomly-picked clones from each of 35 cDNA libraries representing various stages of grain and plant development are being single pass sequenced. The annotated sequence data is being submitted to our, publicly accessible, online database (http://www.cerealsdb.uk.net). The sequencing of cDNAs has been a major component of this project. To date we have sequenced >5000 ESTs. Our protocol can be separated into three main components, template preparation, sequencing reactions and the sequence reading and analysis. Development of these into a reliable and efficient high throughput method has required refinements to each stage, but the most significant area of optimisation has been in generating sequencing templates from bacterial cultures. Template production can be achieved by either plasmid purification methods or directly via PCR from individual bacterial clones. Our progress has made use of both options. However, for reasons of enhanced quality and reliability we have focused on a PCR-based approach. During method developments we have experimented with many aspects of the entire protocol and here will expand upon the optimisations, our conclusions and will describe our final approach.