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A PAC/BAC End-sequence Database for Human Genomic Sequencing

Glen A. Evans, Dave Burbee, Chris Davies, Trey Fondon, Tammy Oliver, Terry Franklin, Lisa Hahner, Shane Probst and Harold R. (Skip) Garner
Genome Science and Technology Center and McDermott Center for Human Growth and Development, The University of Texas Southwestern Medical Center at Dallas.

While current plans call for completing the human genome sequence in 2003, major obstacles remain in achieving the speed and efficiency necessary to complete the task of mapping and sequencing. We proposed a novel approach to large scale construction of sequence-ready physical clone maps of the human genome utilizing end-specific sequence sampling. A earlier pilot project was initially carried out to develop a GSS (genomic sequence sampled) map of human chromosome 11 by sequencing the ends of 17,952 chromosome 11 specific cosmids. This chromosome 11-specific end-sequence database allows rapid and sensitive detection of clone overlaps for chromosome 11-sequencing. In this DOE funded pilot project, we proposed a effort to evaluate the utility of PAC and BAC end-sequences representing the entire human genome as a tool for complete, high accuracy mapping and sequencing. We utilized total genomic PAC/BAC libraries constructed by P. de Jong, RPCI, followed by end-sequencing of both ends of each clone in the library and limited regional mapping of a subset of clones as sequencing nucleation points by FISH (Fluorescence in situ hybridization). To initiate regional analysis, a single clone was be sequenced by shotgun or primer directed sequencing, the entire sequence used to search the end-database for overlapping clones, and the minimal overlapping clones for extending the sequence selected. We demonstrated that this approach allows a rational and efficient simultaneous mapping and sequencing of portions of the genome, as well as expediting the coordination and exchange of information between large and small groups participating in the human genome project. This pilot project involved automated end-sequencing of approximately 5000 PAC and BAC clones representing portions of chromosome 11 as well as portions of the entire human genome. The clones and resulting end-sequence data base were evaluated for their use in 1) nucleating regions of interest for large scale sequencing concentrating on regions of chromosome 11, 2) the evaluation of mapping accuracy and integrity and 3) the evaluation of random clone end sequence libraries. A technique for high throughput DNA sequencing was developed using a Beckman/Sagian robotic system, ABI 377 automated sequencers and automated sequence data processing, annotation. FISH analysis of a sample of over 200 PAC clones was carried out and to define the chimera rate in existing PAC libraries. Continuation of this project would include PAC and BAC end-sequencing of a sufficient number of clones to represent the entire human genome. However, with the premature termination of this pilot project, current goals are directed towards completion of the work underway and preparing data for publication.

Last modified: Wednesday, October 22, 2003

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