Introduction to the Workshop
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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.
BUILDING A PHYSICAL MAP BY STS CONTENT MAPPING TO SUPPORT LARGE SCALE GENOMIC SEQUENCING: THE DROSOPHILA GENOME PROJECT
William J. Kimmerly, Karen E. Stultz, Keith Lewis, Veronica M. Lustre, Dazhong Sun, Christopher H. Martin, and Michael J. Palazzolo.
Human Genome Center, MS 74-157, Lawrence Berkeley Laboratory, 1 Cyclotron Rd., Berkeley, CA 94720
We are constructing a bacteriophage P1-based physical map of the euchromatic genome of Drosophila melanogaster. The ultimate goals of our physical mapping project are to construct a physical map annotated with genetic and biological information, and to identify a minimal number of P1 genomic clones from the library that represent the genome as sequencing templates for the directed genomic sequencing project at LBL. The mapping approach utilizes sequence-tagged sites, or STS markers which fall into three classes: sequences from the ends of P1 genomic inserts, genomic sequences flanking rescued P elements from 2nd and 3rd chromosomal lethals, and known Drosophila genes. The major class of STS markers derive the ends of genomic inserts of individual clones in the library and are obtained by direct sequencing of full-length P1 genomic clones. The mapping strategy we are pursuing, referred to as double-end clone-limited, emphasizes the importance and utility of STS markers derived from the ends of clones in the library. This mapping strategy allows the assignment of all clones in the library to contigs using the fewest number of markers and generates contigs larger than a random or single-end approach.
For these experiments we are using a five-hit P1 genomic library (99% statistical coverage). Using the unique sequences defined as STS markers, primers are designed for use in a PCR-based mapping strategy. STS markers are mapped in the library by screening pools of P1 DNA by PCR in a two-tiered scheme. These mapping data are used to construct contigs of overlapping clones and to determine linkage of adjacent STS markers. Thus far we have mapped over 1500 STS markers. So far nearly 400 contigs have been constructed which together cover over 100 megabases, or about 75% of the euchromatic genome. Our goal at LBL is to map a total of 3000 STS markers towards a preliminary physical map by July 1995, at which time the clone-limited phase of the mapping project should be complete, and all clones in the library should be assigned to contigs. The next stage of the physical mapping project will attempt to join adjacent contigs into larger ones.
We have successfully built two multi-clone contigs of 350-400 kb that served as our initial targets for large-scale genomic sequencing. These targets were the Bithorax complex (BX-C) and the Antennapedia complex (ANT-C). Both complexes have been nearly completely sequenced and submitted to Genbank by the LBL directed genomic sequencing group. In collaboration with Michael Ashburner and colleagues in Cambridge, England we have focused much of our mapping effort on a 2 megabase region of chromosome 2 encompassing the polytene bands 34D-36A. This region contains the well-studied Adh gene, and is rich in genetic information with numerous chromosomal breakpoints, P element insertions, and lethal complementation groups. This region is now covered by a small number of large contigs and many P1 clones from this region are in the process of being sequenced at LBL.
The Drosophila Genome Project is a collaboration between the biology, informatics, and engineering groups at the LBL Human Genome Center. The project also includes Gerald Rubin of the University of California, Berkeley, Allan Spradling of the Carnegie Institute in Baltimore, MD who is contributing the genetic verification of P element lethal lines used in the project, and Dan Hartl of Harvard University in Cambridge, MA who has provided in situ hybridization data for over 2500 P1 clones in the library being used for STS generation.