Plant & Animal Genome IX Conference

Poster: Sequencing & EST
P26

PRODUCTION OF EST TOOLS FOR BARLEY GENE DISCOVERY AND EXPLOITATION

Plant Genomics Laboratory, Institute of Biotechnology, University of Helsinki, P.O. Box 56, FIN-00014 Helsinki, Finland

Barley is the major crop in both cultivated area and total yield in Finland. As part of our research program including retrotransposon-based molecular marker development, we have undertaken an EST project based on a two-rowed Finnish malting variety, Saana. Libraries have been constructed of germinating embryos, roots, shoots, and cell cultures in the Stratagene Unizap XR vector. PCR and sequencing reactions are set up on robotic workstations and sequencing is done on a 96-capillary device (ABI Prism 3700). We currently have the capacity to sequence ~500 clones a day. As of 1.10.2000, we have produced 7681 sequences >300 nt, falling into 4659 clusters, 70% of which give a database hit. To cope with the rapid accumulation of sequence data, we have developed an integrated informatics system based on freely available software and custom-made Java components. The System is running on a Linux server. This design philosophy has made the system cheap, effective and easily ported and adapted to similar projects. Two other EST sequencing projects at the University of Helsinki are already using the system. ESTs are clustered based on sequence homology with the CAP2 algorithm. We also do a homology search against sequence databases with Blast2. All data from the various components is then processed, filtered and entered in AceDB database. While the AceDB software itself suits our purposes well, we found it necessary to completely re-write the DB model. We are currently applying semi-automated annotation protocols to help with this enormous task.