Lawrence Livermore National Laboratory
Human Genome Center
lieu of individual abstracts, research projects and investigators at LLNL
Human Genome Center are represented in this narrative. More information
can be found on the center's Web
In the past 2 years, the center's goals have undergone an exciting evolution. This change is the result of several factors, both intrinsic and extrinsic to the Human Genome Project. They include: (1) successful completion of the center's first-phase goal, namely a high-resolution, sequence-ready map of human chromosome 19; (2) advances in DNA sequencing that allow accelerated scaleup of this operation; and (3) development of a strategic plan for LLNL's Biology and Biotechnology Research Program that will integrate the center's resources and strengths in genomics with programs in structural biology, individual susceptibility, medical biotechnology, and microbial biotechnology.
Center Organization and Activities
The sequencing group is divided into several subprojects. The core team is responsible for the construction of sequence libraries, sequencing reactions, and data collection for all templates in the random phase of sequencing. The finishing team works with data produced by the core team to produce highly redundant, highly accurate "finish" sequence on targets of interest. Finally, a team of researchers focuses specifically on development, testing, and implementation of new protocols for the entire group, with an emphasis on improving the efficiency and cost basis of the sequencing operation.
The functional genomics team is responsible for assembling and characterizing clones for the Integrated Molecular Analysis of Gene Expression (called IMAGE) Consortium and cDNA sequencing, as well as for work on gene expression and comparative genomics. The effort emphasizes genes involved in DNA repair and links strongly to LLNL's gene-expression and structural biology efforts. In addition, this team is working closely with Oak Ridge National Laboratory (ORNL) to develop a comparative map and the sequence data for mouse regions syntenic to human chromosome 19.
Informatics and Analytical Genomics
The center interacts extensively with other efforts within the LLNL Biology and Biotechnology Research Program and with other programs at LLNL, the academic community, other research institutes, and industry. More than 250 collaborations range from simple probe and clone sharing to detailed gene family studies. The following list reflects some major collaborations.
The LLNL Human Genome Center has excelled in several areas, including comparative genomic sequencing of DNA repair genes in human and rodent species, construction of a metric physical map of human chromosome 19, and development and application of new biochemical and mathematical approaches for constructing ordered clone maps. These and other major accomplishments are highlighted below.
Genomic sequencing currently is the dominant function of Livermore's
Human Genome Center. The physical mapping effort will ensure an ample supply
of sequence-ready clones. For sequencing targets on chromosome 19, this
includes ensuring that the most stable clones (cosmids, BACs, and PACs)
are available for sequencing and that regions with such known physical
landmarks as STSs and expressed sequenced tags (ESTs) are annotated to
facilitate sequence assembly and analysis. The following targets are emphasized
for DNA sequencing:
The instrumentation effort has three major thrusts: (1) continued development or implementation of laboratory automation to support highthroughput sequencing; (2) development of the next-generation DNA sequencer; and (3) development of robotics to support highdensity BAC clone screening. The last two goals warrant further explanation.
The new DNA sequencer being developed under a grant from the National Institutes of Health, with minor support through the DOE genome center, is designed to run 384 lanes simultaneously with a low-viscosity sieving medium. The entire system would be loaded automatically, run, and set up for the next run at 3-hour intervals. If successful, it should provide a 20- to 40-fold increase in throughput over existing machines.
An LLNL-designed high-precision spotting robot, which should allow a density of 98,304 spots in 96 cm2, is now operating. The goal of this effort is to create highdensity filters representing a 10X BAC coverage of both human and mouse genomes (30,000 clones = 1X coverage). Thus each filter would provide ~3X coverage, and eight such filters would provide the desired coverage for both genomes. The filters would be hybridized with amplicons from individual or regionspecific cDNAs and ESTs; given the density of the BAC libraries, clones that hybridize should represent a binned set of BACs for a region of interest. These BACs could be the initial substrate for a BAC sequencing strategy. Performing hybridizations in parallel in mouse and human DNA facilitates the development of the mouse map (with ORNL involvement), and sequencing BACs from both species identifies evolutionarily conserved and, perhaps, regulatory regions.
Information generated by sequencing human and mouse DNA in parallel is expected to expand LLNL efforts in functional genomics. Comparative sequence data will be used to develop a high-resolution synteny map of conserved mouse-human domains and incorporate automated northern expression analysis of newly identified genes. Long range, the center hopes to take advantage of a variety of forms of expression analysis, including site-directed mutation analysis in the mouse.
The Livermore Human Genome Center has undergone a dramatic shift in emphasis toward commitment to largescale, highaccuracy sequencing of chromosome 19, other chromosomes, and targeted genomic regions in the human and mouse. The center also is committed to exploiting sequence information for functional genomics studies and for other programs, both in house and collaboratively.
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