Human Genome Center 
Lawrence Livermore National Laboratory 
Biology and Biotechnology 
Research Program 
7000 East Avenue, L452 
Livermore, CA 94551 

Anthony V. Carrano 
Director 
510/422-5698, Fax: /423-3110 
carrano1@llnl.gov 

Linda Ashworth 
Assistant to Center Director 
510/422-5665, Fax: -2282 
ashworth1@llnl.gov 
 
 

In 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 site. 
 
 
 
 
 

Chromosome 19 Map  (34k JPG)

 
 

The Human Genome Center at Lawrence Livermore National Laboratory (LLNL) was established by DOE in 1991. The center operates as a multidisciplinary team whose broad goal is understanding human genetic material. It brings together chemists, biologists, molecular biologists, physicists, mathematicians, computer scientists, and engineers in an interactive research environment focused on mapping, DNA sequencing, and characterizing the human genome. 

Goals and Priorities 

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. 

The primary goal of LLNL's Human Genome Center is to characterize the mammalian genome at optimal resolution and to provide information and material resources to other in-house or collaborative projects that allow exploitation of genomic biology in a synergistic manner. DNA sequence information provides the biological driver for the center's priorities: 

• Generation of highly accurate sequence for chromosome 19. 
• Generation of highly accurate sequence for genomic regions of high biological interest to the mission of the DOE Office of Biological and Environmental Research (e.g., genes involved in DNA repair, replication, recombination, xenobiotic metabolism, and cell-cycle control). 
• Isolation and sequence of the full insert of cDNA clones associated with genomic regions being sequenced. 
• Sequence of selected corresponding regions of the mouse genome in parallel with the human. 
• Annotation and position of the sequenced clones with physical landmarks such as linkage markers and sequence tagged sites (STSs). 
• Generation of mapped chromosome 19 and other genomic clones [cosmids, bacterial artificial chromosomes (BACs), and P1artificial chromosomes (PACs)] for collaborating groups. 
• Sharing of technology with other groups to minimize duplication of effort. 
• Support of downstream biology projects, for example, structural biology, functional studies, human variation, transgenics, medical biotechnology, and microbial biotechnology with knowhow, technology, and material resources. 

Center Organization and Activities 

Completion and publication of the metric physical map of human chromosome 19 (pictured at left)  in 1995 has led to consolidation of many functions associated with physical mapping, with increased emphasis on DNA sequencing. The center is organized into five broad areas of research and support: sequencing, resources, functional genomics, informatics and analytical genomics, and instrumentation. Each area consists of multiple projects, and extensive interaction occurs both within and among projects. 

Resources 
The resources group provides mapped clonal resources to the sequencing teams. This group performs physical mapping as needed for the DNA sequencing group by using fingerprinting, restriction mapping, fluorescence in situ hybridization, and other techniques. A small mapping effort is under way to identify, isolate, and characterize BAC clones (from anywhere in the human genome) that relate to susceptibility genes, for example, DNA repair. These clones will be characterized and provided for sequencing and at the same time contribute to understanding the biology of the chromosome, the genome, and susceptibility factors. The mapping team also collaborates with others using the chromosome 19 map as a resource for gene hunting. 

Mouse-Human  Similarities (33k JPG)

 
 
 
  

Researching  Human-Mouse  Homologies (20k JPG)

 
 
 
 
 
 
 
 
  

Comparative Sequencing (19k JPG)

 

Informatics and Analytical Genomics 
The informatics and analytical genomics group provides computer science support to biologists. The sequencing informatics team works directly with the DNA sequencing group to facilitate and automate sample handing, data acquisition and storage, and DNA sequence analysis and annotation. The analytical genomics team provides statistical and advanced algorithmic expertise. Tasks include development of modelbased methods for data capture, signal processing, and feature extraction for DNA sequence and fingerprinting data and analysis of the effectiveness of newly proposed methods for sequencing and mapping. 

Instrumentation 
The instrumentation group also has multiple components. Group members provide expertise in instrumentation and automation in highthroughput electrophoresis, preparation of high-density replicate DNA and colony filters, fluorescence labeling technologies, and automated sample handling for DNA sequencing. To facilitate seamless integration of new technologies into production use, this group is coupled tightly to the biologist user groups and the informatics group. 

Collaborations 

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. 

• Integration of the genetic map of human chromosome 19 with corresponding mouse chromosomes (ORNL). 
• Miniaturized polymerase chain reaction instrumentation (LLNL). 
• Sequencing of IMAGE Consortium cDNA clones (Washington University, St.Louis). 
• Mapping and sequencing of a gene associated with Finnish congenital nephrotic syndrome (University of Oulu, Finland).   

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. 

• Completion of highly accurate sequencing totaling 1.6 million bases of DNA, including regions spanning human DNA repair genes, the candidate region for a congenital kidney disease gene, and other regions of biological interest on chromosome 19. 
• Completion of comparative sequence analysis of 107,500 bases of genomic DNA encompassing the human DNA repair gene ERCC2 and the corresponding regions in mouse and hamster. In addition to ERCC2, analysis revealed the presence of two previously undescribed genes in all three species. One of these genes is a new member of the kinesin motor protein family. These proteins play a wide variety of roles in the cell, including movement of chromosomes before cell division. 
• Complete sequencing of human genomic regions containing two additional DNA repair genes. One of these, XRCC3, maps to human chromosome 14 and encodes a protein that may be required for chromosome stability. Analysis of the genomic sequence identified another kinesin motor protein gene physically linked to XRCC3. The second human repair gene, HHR23A, maps to 19p13.2. Sequence analysis of 110,000 bases containing HHR23A identified six other genes, five of which are new genes with similarity to proteins from mouse, human, yeast, and Caenorhabditis elegans. 
• Complete sequencing of full-length cDNAs for three new DNA repair genes (XRCC2, XRCC3, and XRCC9) in collaboration with the LLNL DNA repair group. 
• Generation of a metric physical map of chromosome 19 spanning at least 95% of the chromosome. This unique map incorporates a metric scale to estimate the distance between genes or other markers of interest to the genetics community. 
• Assembly of nearly 45 million bases of EcoRI restriction-mapped cosmid contigs for human chromosome 19 using a combination of fingerprinting and cosmid walking. Small gaps in cosmid continuity have been spanned by BAC, PAC, and P1 clones, which are then integrated into the restriction maps. The high depth of coverage of these maps (average redundancy, 4.3-fold) permits selection of a minimum overlapping set of clones for DNA sequencing. 
• Placement of more than 400 genes, genetic markers, and other loci on the chromosome 19 cosmid map. Also, 165 new STSs associated with pre-mapped cosmid contigs were generated and added to the physical map. 
• Collaborations to identify the gene (COMP) responsible for two allelic genetic diseases, pseudoachondro-plasia and multiple epiphyseal dysplasia, and the identification of specific mutations causing each condition. 
• Through sequence analysis of the 2A subfamily of the human cytochrome P450 enzymes, identification of a new variant that exists in 10% to 20% of individuals and results in reduced ability to metabolize nicotine and the antiblood-clotting drug Coumadin. 
• Location of a zinc finger gene that encodes a transcription factor regulating blood-cell development adjacent to telomere repeat sequences, possibly the gene nearest one end of chromosome 19. 
• Completion of the genomic and cDNA sequence of the gene for the human Rieske FeS protein involved in mitochondrial respiration. 
• Expansion of the mouse-human comparative genomics collaboration with ORNL to include study of new groups of clustered transcription factors found on human chromosome 19q and as syntenic homologs on mouse chromosome 7. 
• Numerous collaborations (in particular, with Washington University and Merck) continuing to expand the LLNL-based IMAGE Consortium, an effort to characterize the transcribed human genome. The IMAGE clone collection is now the largest public collection of sequenced cDNA clones, with more than one million arrayed clones, 800,000 sequences in public databases, and 10,000 mapped cDNAs. 
• Development and deployment of a comprehensive system to handle sample tracking needs of production DNA sequencing. The system combines databases and graphical interfaces running on both Mac and Sun platforms and scales easily to handle largescale production sequencing. 
• Expansion of the LLNL genome center's World Wide Web site to include tables that link to each gene being sequenced, to the quality scores and assembled bases collected each night during the sequencing process, and to the submitted GenBank sequence when a clone is completed. 
• Implementation of a new database to support sequencing and mapping work on multiple chromosomes and species. Web-based automated tools were developed to facilitate construction of this database, the loading of over 100 million bytes of chromosome 19 data from the existing LLNL database, and automated generation of Webbased input interfaces. 
• Significant enhancement of the LLNL Genome Graphical Database Browser software to display and link information obtained at a subcosmid resolution from both restriction map hybridization and sequence feature data. Features, such as genes linked to diseases, allow tracking to fragments as small as 500 base pairs of DNA. 
• Development of advanced micro-fabrication technologies to produce electrophoresis microchannels in large glass substrates for use in DNA sequencing. 
• Installation of a new filter-spotting robot that routinely produces 6 X 6 X 384 filters. A 16 X 16 X 384 pattern has been achieved.
• Upgrade of the Lawrence Berkeley National Laboratory colony picker using a second computer so that imaging and picking can occur simultaneously. 

Future Plans 

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: 
 

• Regions of high gene density, including regions containing gene families. 
• Chromosome 19, of which at least 42 million bases are sequence ready. 
• Selected BAC and PAC clones representing regions of about 0.2 million to 1 million bases throughout the human genome; clones would be selected based on such high-priority biological targets as genes involved in DNA repair, replication, recombination, xenobiotic metabolism, cell-cycle checkpoints, or other specific targets of interest. 
• Selected BAC and PAC clones from mouse regions syntenic with the genes indicated above. 
• Full-insert cDNAs corresponding to the genomic DNA being sequenced. 
The informatics team is continuing to deploy broader-based supporting databases for both mapping and sequencing. Where appropriate, Web and Java-based tools are being developed to enable biologists to interact with data. Recent reorganization within this group enables better direct support to the sequencing group, including evaluating and interfacing sequence-assembly algorithms and analysis tools, data and process tracking, and other informatics functions that will streamline the sequencing process. 

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. 

Summary 

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.