Human Genome Project Information Archive
1990–2003

Sequencing at Other Institutions

New Strategies, Resources Reported

Report from 1999 DOE Genome Meeting

The exciting potential of high-throughput sequencing was evident as presenters detailed progress in effective strategies for handling repeat telomeric regions, high-speed DNA analysis systems, and new vectors.

Verifying Sequence of Subtelomeric Regions
Producing correct maps and sequences in human DNA regions containing a high percentage of repetitive sequences is a challenge. Particularly troublesome are the chromosome ends (telomeres), which can have complex arrays of repeats. The subtelomeric region of human chromosome 7q was sequenced largely at Los Alamos National Laboratory while Bob Moyzis was director of the Center for Human Genome Studies. At the Oakland meeting, Moyzis [now at University of California (UC), Irvine] reported a quality-control analysis using the RARE (for RecA-Assisted Restriction Endonuclease) cleavage technology.

Linda Ashworth (LLNL) and Han-Chang Chi (University of California, Irvine)

This technology allows restriction maps to be constructed directly from genomic DNA rather than from DNA clones that are more prone to rearrangements. Using DNA from PCR products generated on genomic DNA, the UC researchers also resequenced about 18% of the 0.13-Mb 7q telomere. These methods confirmed prior mapping and sequencing results. The group is completing the mapping and sequencing of two additional telomeres, 9q and 11q, and next will target telomeric regions of chromosomes 5, 16, and 19.

Microfabricated Devices
Microfabrication of devices for DNA sample preparation, electrophoretic separation, and detection is advancing a new generation of high-speed DNA analysis systems. Microdevices require less sample input, present narrower sample zones, generate less heat during electrophoresis, and thus suffer less sample-diffusion broadening during shorter, more effective run times. Although some of these devices will be used to finish the Human Genome Project, most will be deployed into the next century as the number of sequencing projects increases. Richard Mathies (UC, Berkeley) previously developed a capillary array electrophoresis device in which DNA was separated in individual glass capillaries, a prototype that has since been commercialized successfully. Sequencing throughput had been limited by the number of capillaries that could fit into an array and by the inefficiency of sample injection. At the Oakland meeting, Mathies described a new microfabricated sequencing device that promises higher throughput (see "New Device Speeds Sequencing"). Detection in the new device is accomplished by the confocal fluorescence optical system used in Mathies' previous technologies, but the group is moving to other detection paradigms. Mathies and colleague Indu Kheterpal reviewed several approaches to using capillary arrays for high-throughput DNA sequencing [see Anal. Chem. 71(1), 31-37A (1999)].

BAC Update: New Vectors, Sequencing Progress
Because of their stability and large insert size (average, 150 kb), BAC clones have played an increasingly important role in human and mammalian genomics in recent years. BAC libraries are being used or developed for almost every extensively characterized genome.

At the Oakland meeting, Melvin Simon (California Institute of Technology) talked about new BAC vectors for the genome project and for tagging BAC clones with gene information to enhance research into gene function in animals and plants. The number of human BAC clones generated is now close to one million. Simon's team has been annotating the "D" library [used for BAC-end sequencing at The Institute for Genomic Research (TIGR) and the University of Washington (UW, see "Gene Riches of Chromosome 19 Revealed")] with ESTs obtained from I.M.A.G.E. Consortium resources [http://www.imageconsortium.org/].

Simon also reported the development of a new BAC vector and an improved method of constructing BAC libraries. The group has begun constructing a series of BAC libraries with much larger insert sizes (182 to 202 kb) for sequencing projects on humans and other organisms, including Arabidopsis, maize, and rice. The larger-insert libraries will provide significant improvement, he noted, to applications in physical mapping, positional cloning, and DNA sequencing.

Pieter de Jong (now at Parke-Davis Laboratory) also described new approaches to library construction and the preparation of new BAC vectors for BAC cloning and transformation-associated recombination (TAR) cloning. (See Web site for more information). When comparisons of multiple gene alleles are desirable, scientists use the TAR strategy invented by Natalay Kouprina and Vladimir Larionov (both at NIH National Institute of Environmental Health Sciences). TAR provides for an economical, selective recloning of target genomic DNA segments. The TARBAC variant has been used to prepare BAC libraries for several less-complex genomes of unicellular eukaryotes to model future work with mammalian TARBAC libraries.

UW and TIGR are generating sequence reads from BAC ends. These sequence tagged connectors (STCs) are useful markers that speed human genome contig building and sequencing. A target of one STC for every 3 kb of the human genome will eliminate bottlenecks in contig development. Beyond use in human genome sequencing, the characterized clones provide excellent resources for biological studies. The STC resource is available on the Web for use by any researcher for clone or sequencing target selection, and the clones can be ordered from commercial resources.

BAC-End Project Web Sites

• Human BAC-End Sequencing: www.ornl.gov/meetings/bacpac.pdf
• University of Washington, Seattle
• TIGR: http://www.tigr.org/tdb/humgen/bac_end_search/bac_end_intro.html

Plasmid Vectors
John Dunn (Brookhaven National Laboratory) and his team have developed improved biochemistry and vectors for the "deletion-factory" approach to sequencing. This involves generating a set of nested deletions from a common parent plasmid and recognizing descendants with progressively shorter inserts. Those chosen for successive sequencing differ in length by less than one sequencing read, and assembly problems are negligible compared to those obtained with shotgun sequencing. The deletion factory approach is particularly useful for regions having numerous repeats that cause troublesome assembly ambiguities during shotgun sequencing.

The new "pZIP" plasmid vectors (named for the way the sequencing progresses along the insert) are maintained in a single-copy state that generally increases the stability of the foreign DNAs they carry and are amplified only when DNA is needed for sequencing. DNA segments up to at least 15 kb have been sequenced, and the reads have been assembled easily. Dunn's team currently is sequencing restriction fragments of human BACs with sizes in the 5- to 15-kb range.

Plasmid Unwinding For Sequencing
Sergei Kozyavkin (Fidelity Systems, Inc.) presented a poster describing applications of ThermoFidelase, a heat-stable enzyme that relaxes plasmid DNA supercoils. Upon relaxation at high temperature with a shift to lower temperature, the covalently closed, duplex DNA is forced into a partially denatured configuration. This enables the binding of primers for Sanger sequencing biochemistry. The contaminating fragments of host DNA retain their original duplex configuration and cannot bind primers. Useful Sanger reaction products thus can be generated selectively from templates of ThermoFidelase-treated plasmids, even in the presence of great excesses of fragmented host DNA. This demonstrates that even single-copy plasmids such as BACs can serve as templates to generate good-quality sequencing ladders within the total complement of excess host Escherichia coli DNA. Accordingly, this technology has substantial promise for simplifying several sequencing protocols, including BAC-end sequencing.

The electronic form of the newsletter may be cited in the following style:
Human Genome Program, U.S. Department of Energy, Human Genome News (v10n3-4).