Ung-Jin Kim, Steve Marsh, Ronald Swanson*, and Melvin I. Simon, Division of Biology, 147-75, California Institute of Technology, Pasadena, CA 91125 and Sorel Fitz-Gibbon and Jeffrey Miller Department of Biology and MBI, University of California, Los Angeles, Los Angeles, CA 90024.
A random shotgun approach to sequencing approximately 1.8 Mb genome of the hyperthermophilic Archaea Pyrobaculum aerophilum has been undertaken. The genomic DNA of this microbe was sheared by sonication and 2-3 kb fragments were subcloned into pUC18 vectors for sequencing. Tile genome was also cloned in the Fosmid vector that permits stable propagation of cosmid-sized Pyrobaculum genomic DNA fragments which are unstable in high copy number cosmid vectors. The Fosmid library with 768 clones, or roughly l5X genomic coverage, was used to rapidly assemble a physical contig map of this organism using more than 100 probes as markers and restriction fingerprint analysis techniques. We are also planning to determine the end sequences of the Fosmid clones. The library and the physical map will eventually be used for gap closure at the final stage of sequence assembly.
Large numbers of sequencing templates are prepared by using an Autogen 740 miniprep machine, and are being sequenced at the Caltech sequencing core facility. Automated sequencing machines are being used to generate sequences from both ends of the template with an average of 500-600 bases of clean sequence per read. With the use of new TaqI polymeraseFS, we expect that the read length as well as the throughput will increase at least by 10-20%. As of October 1, 1995, we have obtained nearly 6,000 reads, and we expect the number will reach 25,000 or roughly 7X genomic covergage by early 1996. The sequences will then be assembled by using TIGR_ASSEMBLER and other commercially available packages, annotated, and will be deposited to publicly accessible repositories.
*Recombinant Biocatalysis, Inc., Kennett Square, PA 19348.
Abstracts scanned from text submitted for January 1996 DOE Human Genome Program Contractor-Grantee Workshop.