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The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.

Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.

Fingerprinting, clustering and sequence recognition by hybridization to short oligomers: informational support for targeted cDNA and genomic sequencing

Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory, Argonne, Illinois 60439-4833
e-mail: milosav@anl.gov
[1]Hyseq Inc., Synnyvalle, California

A novel approach for sequence recognition has been developed and successfully tested: lists of complementary short oligomers that have been identified in a clone by hybridization to a few hundred short oligomer probes have for the first time been used to correctly identify matches in DNA-sequence databases; alternatively, known DNA sequences have also been used to correctly identify matches among tens of thousands of clones. In addition to the comparisons against known sequences, clones have also been mutually compared based on their hybridization fingerprints in order to identify clone identities, homologies, and overlaps. Clustering of clones based on their hybridization fingerprints and sequence recognition based on oligomer lists provide valuable targeting information for both cDNA and genome sequencing.

A data production line for the hybridization of hundreds of thousands of clones against hundreds of short oligomers (developed by R. Drmanac et al.) has been applied to obtain tens of millions of clone/probe scores. Informatics has been developed for experiment planning, data archiving, and data analysis.

In a model cDNA experiment, 29,500 clones from human brain cDNA libraries (prepared by B. Soares et al.) were spotted in duplicate on 11 different filters containing 3456, 7776, or 31,104 dots. The filters were probed with 107-215 decamer probes containing 6-8 nondegenerate contiguous nucleotide positions. Clustering based on hybridization signatures revealed 16,750 clusters representing distinct gene sequences. Representative clones from the 100 largest clusters were chosen for comparison against a database consisting of 195 DNA sequences of genes that were expected to be highly expressed in brain; comparisons revealed 6 identities and 5 close homologies that were subsequently confirmed by gel sequencing.

In a model genomic DNA experiment, 15,000 genomic 2-kb clones from E. coli and 15,000 clones from a few related bacteria were spotted in duplicate on 2 filters containing 31,104 dots each. In addition to bacterial clones, a number of control clones of known sequence from a human intron were spotted. One of the filters was hybridized with 478 probes while hybridizations with the other are still in progress. Clustering of the first filter revealed 4500 groups of clones corresponding to distinct genomic regions, as well as distinct groups corresponding to control clones. By taking a single representative from each cluster, a database consisting of 4500 clones that are expected to cover the complete E. coli genome, plus the control clones, was constructed. The known sequences of 8 control clones were then used in a search against 4500 oligomer lists; most searches correctly identified identical or highly overlapping control clones. Similar E. coli sequence recognition experiments are in progress. Methods are being developed for clone ordering based on hybridization fingerprints.

This work is supported by U.S. Department of Energy, Office of Health and Environmental Research, under Contract W-31-109-Eng-38.