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Human Genome News, May 1991; 3(1)
Physical Mapping Efforts Going Well; Gels Increasing Sequencing Efficiency The DOE Human Genome Program held its second Contractor-Grantee Workshop in Santa Fe, New Mexico, on February 17-20. More than 200 program-sponsored scientists attended the meeting, in addition to invited guests and industry representatives. DOE-supported human genome research projects are conducted at 7 DOE national laboratories (including its 3 human genome centers), 37 major universities, and 32 companies through collaborations and awards. Projects were represented by oral presentations or posters.
Six platform sessions focused on the following:
David Galas, Associate Director, Office of Health and Environmental Research (OHER), spoke about the relationship between the Human Genome Program and other OHER programs. Michael Yesley [Los Alamos National Laboratory (LANL)] presented ethical, legal, and social issues pertaining to data produced in the genome project.
The general impression conveyed by most presenters was that physical mapping efforts are going well; chromosomes 16, 19, and portions of 11 are now well covered with large numbers of assembled contigs. Fluorescence in situ hybridization (FISH) is emerging as an extremely effective method for ordering cloned probes.
Fractionating DNA fragments is becoming much faster with capillary or thin gel slab electrophoresis. Informatics support for most physical mapping efforts and for large-scale DNA sequencing remains problematic, and more focus is needed on the immediate informatics needs of ongoing biology projects.
Many parallel efforts under way in cloning, informatics, mapping, and sequencing will further improve the technologies required for genomics. Program participants feel that this situation is healthy at present and that a few approaches will emerge as those most likely to accelerate the project.
One theme that recurred frequently during the 3-day meeting was the rapid change in bottlenecks or rate-limiting steps, particularly in DNA-sequencing efforts. Advances in raw sequencing speed make automated production of DNA samples and melding of raw sequence reads increasingly important.
Generally, impressive progress continues in the construction of physical maps of selected human chromosomes. Yeast artificial chromosomes (YACs), though not without problems, are proving helpful in linking cosmid contigs or providing rapid initial coverage of a region.
The powerful new FISH technique is providing mapping data at several levels of resolution, extending from metaphase chromosomes. It offers a rapid and accurate method of regional clone assignment to a chromosome band. Barbara Trask [Lawrence Livermore National Laboratory (LLNL)] showed that much higher resolution mapping can be achieved with interphase cells; once markers are known to be close, their relative order can be determined and distances estimated in the 50-kb to 1-Mb range.
The presence of a selectable marker near the long-arm telomere of chromosome 16 made possible the construction of a set of hybrid cell lines particularly convenient for that chromosome. Grant Sutherland (Adelaide Children's Hospital, South Australia) explained this method and showed the usefulness of FISH in characterizing chromosomal rearrangements.
Glen Evans (Salk Institute) and collaborators have used a combination of methods in mapping chromosome 11. In constructing several multimegabase contigs of cloned DNA segments, Evans used a YAC as a hybridization probe against a filter array of cosmids to identify covered cosmids. Given the appropriate libraries, a series of overlapping clones for 1- to 2-Mb regions apparently can be isolated in a matter of weeks.
Both the chromosome 19 program at LLNL and that of chromosome 16 at LANL have collected about two-thirds of their chromosomes into cosmid contigs. A variety of very effective automated approaches are being used to expand and link these contigs. Existing maps are dense enough to be useful to investigators wishing to locate genes on these chromosomes; about 80 of randomly picked probes will fall on contigs or clones already mapped. To aid such studies, a series of anchor points has been established between the current genetic and physical maps. No clone-order discrepancies are evident between the two map types for either chromosome.
Excellent progress is being made with maps of X-chromosome regions. Thomas Caskey, David Nelson, and their coworkers (Baylor College of Medicine) are focusing on several areas that contain genes of interest rather than attempting to construct a complete map of this large chromosome. Caskey described the potency of using tandem simple sequence repeats as genetic markers.
In the next few years, nearly complete contig maps should be obtained on chromosomes 11, 16, 19, and X.
Several groups reported new approaches toward large-insert cloning vectors:
Gary Hermanson (Glen Evans' laboratory, Salk Institute) gave an interesting presentation on isolating the ends of YAC inserts by homologous recombination, a promising technique for walking within YAC libraries. Sherman Weissman (Yale University) reported on his group's efforts to achieve normalized cDNA libraries from human thymus. The research is proceeding well in setting the stage for large-scale sequence analysis of the corresponding cDNAs from various tissues.
A goal of the Human Genome Project is to reduce DNA sequencing costs. While considerable attention has been given to data-collection instrumentation and data-analysis hardware and software, the scientists felt that more emphasis is needed on technologies that will reduce costs by increasing throughput and template quality.
A number of presentations dealt with transposon insertions to assist in DNA sequencing, thereby minimizing redundancy associated with shotgun cloning strategies. Douglas Berg (Washington University School of Medicine) and his group have exploited the bacterial Tn5supF transposon system by inserting a known 260-bp sequence into lambda clones to serve as the priming site for sequencing. This procedure eliminates the need to subclone large inserts, since sequencing could be accomplished directly from the lambda clone by primer walking from the inserted transposon. Claire Berg (University of Connecticut) described the use of transposon (yd)(Tn1000) to sequence plasmid inserts. Robert Weiss and Raymond Gesteland (University of Utah) reported on applying this method to sequence plasmids containing 10-kb inserts. Their clone-pooling scheme is employed for rapid mapping of the transposon integration sites; the mapped transposons are then used as primer sites for multiplexed dideoxy sequencing.
William Studier (Brookhaven National Laboratory) reported on priming DNA sequencing reactions by using sequences within the insert itself. George Church (Harvard Medical School) presented the status of his developments for computer-assisted multiplexed sequencing. Arthur Riggs (Beckman Research Institute) described a ligation-mediated genomic sequencing strategy that allows sequence information to be derived from genomic DNA without subcloning.
Bruce Jacobson (Oak Ridge National Laboratory) and Heinrich Arlinghaus (Atom Sciences, Inc.) are implementing tin, iron, and lanthanide isotopes as reporter groups for DNA sequencing. Sputter-Initiated Resonance Ionization Spectroscopy or Laser Atomization Resonance Ionization Spectroscopy (two forms of mass spectrometry used to assay these reporter groups), appear to have striking sensitivity and speed of analysis. The thin-layer capillary electrophoresis techniques reported by Barry Karger (Northeastern University) present notable opportunities for speeding up throughput in the analysis of DNA sequence fragments. The impressive data of Lloyd Smith (University of Wisconsin) suggested that thin-layer capillary gels will work effectively in sequencingfragment separation and provide multiple lane parallelism for DNA sequence analysis. The use of exonucleases and the ability to detect single nucleotides for the analysis of single DNA molecules are being advanced by Richard Keller's (LANL) group. (See associated article.)
Several presentations were made on scanning tip microscopy. An outstanding one by Rodney Balhorn and Wigbert Siekhaus (LLNL) used scanning tunneling microscopy to reveal images of monolayers of adenine and thymine bases that are consistent with the known physical structure of these molecules.
Other presenters described methods for detecting DNA for either sequencing or mapping. Christopher Martin and Irena Bronstein (Tropix, Inc.) demonstrated the application of chemiluminescence in Sanger dideoxy sequencing protocols. This technique uses biotinylated primers in standard sequencing reactions. Richard Mathies and Alexander Glazer (University of California, Berkeley) demonstrated analytical miniaturized fractionations of fluorescing DNA fragments under a confocal laser microscope system.
Two novel methods are being developed as third-generation sequencing approaches. Joe Gray (LLNL) and coworkers are exploring the use of X-ray diffraction for DNA sequence analysis. Radoje Drmanac and Radomir Crkvenjakov (Argonne National Laboratory) described sequencing by hybridization: short oligomers (8 to 9 mers) with known sequence are hybridized to short fragments of DNA with unknown sequence. The collection of oligomers that hybridize to a fragment reveal the sequence. The group has successfully sequenced a small DNA segment and is now scaling up for practical applications.
Presentations showed broad progress in applying computer technology to the Human Genome Project. Jim Fickett (LANL) and colleagues noted that the chromosome 16 map requires management of nearly a million data items, the bulk from fingerprinting about 4000 clones. With over 60% of chromosome 16 covered by cosmid contigs, attention is shifting to YACs, FISH, and sequence tagged sites (STSs) for closing intercontig gaps, thereby requiring extensions to the existing data system. Chris Fields (New Mexico State University) and colleagues described an automated system for screening candidate STS sequences and predicting good polymerase chain reaction priming sites on them.
Many posters and one presentation addressed the central computing problem of sequence matching. Eugene Lawler discussed a new dynamic programming method developed with William Chang (both at University of California, Berkeley) that is 4 or 10 times faster (using nucleotide or amino acid sequences, respectively) than the best previous dynamic programming algorithm.
Providing and controlling access to multiple data sources is a key challenge for genomic research. Thomas Slezak (LLNL) reported on some proof-of-concept tests that showed how controlled, multiple database access can be achieved by using client-server architecture found in most commercial, multiuser database systems. Peter Pearson (Johns Hopkins University) described the public database efforts under way with the Genome Data Base (GDB) project (sponsored by the Howard Hughes Medical Institute). He also discussed collaborations with LANL and LLNL for sharing physical mapping data and the possibility of researchers viewing laboratory-generated map data in the information-rich context of GDB.
Charles R. Cantor and HGMIS gratefully acknowledge contributions to this article by Elbert W. Branscomb, Anthony V. Carrano, Leroy E. Hood, Robert K. Moyzis, and Robert J. Robbins.
HGMIS is preparing the 1991 revision of the DOE Human Genome 1989-90 Program Report.
To update the report and accurately reflect DOE-funded genome research projects, HGMIS asks investigators to send abstracts, photographs, and figures. Investigators who have not already submitted this material are reminded to forward it to:
The electronic form of the newsletter may be cited in the following style:
Human Genome Program, U.S. Department of Energy, Human Genome News (v3n1).
The Human Genome Project (HGP) was an international 13-year effort, 1990 to 2003. Primary goals were to discover the complete set of human genes and make them accessible for further biological study, and determine the complete sequence of DNA bases in the human genome. See Timeline for more HGP history.
Published from 1989 until 2002, this newsletter facilitated HGP communication, helped prevent duplication of research effort, and informed persons interested in genome research.
