DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

98. From EST to High Quality cDNA: The BDGP Pipeline for the Construction of Drosophila cDNA Resources

Mark Stapleton¹, Damon Harvey, Peter Brokstein, and Gerald M. Rubin

Berkeley Drosophila Genome Project-University of California, Berkeley, CA and ¹Lawrence Berkeley National Laboratory, Berkeley, CA

staple@bdgp.lbl.gov

The Drosophila Genome Center's future goals are centered around functional genomics. By taking advantage of the Drosophila genomic sequence, we intend to develop tools and technologies for answering biological questions in a high-throughput environment. Our first step in this direction is to create a publicly available unigene set of Drosophila cDNAs and sequence them to high quality.

We have finished the second stage of creating a set of Drosophila cDNAs. The first stage consisted of sequencing greater than 80,000 5' ESTs and was finished March 19, 1999. For the second stage, these ESTs were then clustered based on their 5' ends to reduce redundancy, which resulted in a set of 12,198 clusters. The clone extending most 5' in each cluster has been selected and rearrayed. We have sequenced the 5' and 3' ends of these clones to verify their identity and to further collapse the set on the basis of their 3' identities. We also determined the length of the cDNA insert in each clone so that optimal sequencing strategies can be applied to specific size ranges. Finally, we have performed pilot experiments for full-length sequencing utilizing transposon-based methods that resulted in the completion of 283 high quality cDNAs.