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

19. Library Strategy for Genome Sequencing Projects

William C. Nierman

The Institute for Genomic Research, Rockville, MD 20850

wnierman@tigr.org

Microbial genome sequencing projects at TIGR have been conducted using two-ended clone sequencing primarily from a small 1.5 to 2 kb insert size plasmid library supplemented with sequence reads from both ends of several hundred 15 - 20 kb lambda clones. We have recently implemented a new shotgun library strategy which incorporates sequence reads from both a small 2 kb insert size library and a larger 10 kb insert size plasmid libraries. This strategy has resulted in a dramatic decline in the number of gaps at the end of the random phase of sequencing for which there is no clone coverage, greatly simplifying the process of closure of the genome. Data from several TIGR sequencing projects will be provided to document this conclusion.

BAC based projects for organisms such as the human and mouse are undertaken to minimize the assembly and closure problems of large repeat rich genomes. The BAC libraries supporting these projects were constructed using partial restriction digests to fragment the genomic DNA prior to ligation to the BAC vector. Due to the non-random distribution of restriction sites for any enzyme in genomic DNA libraries thus constructed always have over-representation and underrepresentation of some regions and no coverage of some small fraction of the genome. These regions of no coverage are revealed as gaps in the BAC contig maps produced by analysis of restriction fingerprints of the BAC clones.

In order to develop a resource for providing clone coverage across these gaps in the BAC contig maps for the human and mouse sequencing efforts, we are constructing BAC libraries from random sheared genomic DNA. The targeted insert sizes are 50 and 100 kb. The human libraries are being constructed with donor DNA collected in strict accordance with appropriate informed consent at Celera Genomics (Hamilton Smith). The mouse libraries are being constructed from male C57BL/6J DNA provided by The Jackson Laboratory with appropriate animal committee review at both The Jackson Laboratory and at TIGR. All libraries are being constructed with very narrow insert size cuts to facilitate easy detection of consequential deletions by clone insert size determinations.