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Human Genome News Archive Edition

Human Genome News, Jan.-Feb. 1995; 6(5): 6

LBL Completes 2 Mb of DNA Sequence with Directed Strategy

By early December 1994, the LBL Human Genome Center had completed a total of 2 Mb of genomic sequence from human and Drosophila DNA using a directed strategy. Acting Center Director Mohandas Narla observed that this accomplishment, an important milestone toward cost-effective and accurate large-scale DNA sequencing, validates the DOE strategy of establishing genome facilities in national laboratories where interdisciplinary scientific teams successfully attack large problems.

LBL's directed DNA-sequencing strategy, developed by Michael Palazzolo and Christopher Martin, produces high-resolution physical maps of P1 clones with a small set of standard primer-binding sites every 300 bp. A mapped (ordered) minimum template set is then sequenced. Compared with random strategies, directed sequencing reduces template preparation by tenfold and sequencing by fivefold. Most important, sequence assembly is straightforward because all templates are mapped relative to each other before sequencing. The approach allows investigators to organize the work into six separate modules: physical mapping, high-resolution physical mapping, transposon insertion, sequencing, data assembly, and editing.

Automation.

Joseph Jaklevic leads the LBL instrumentation group in automating the new sequencing strategy as an assembly line of individual steps. These steps include clone picking and arraying, robotic manipulation of libraries, preparation of PCR and sequencing reactions, custom oligonucleotide synthesis of PCR primers, thermal cycling for PCR-amplification reactions, large-scale gel loading and running, image processing, and plasmid preparation.

The instrumentation group has developed modules that are already everyday tools of the sequencing-production group. An automated system captures and analyzes information from agarose gels and automates data acquisition, interpretation, and decision making (e.g., picking valid clones to build a minimum subset), thus reducing fragment-sizing time from 8 h to between 4 and 8 min. A robotics-compatible, multistation thermal cycler is now performing 600 PCR amplifications/h in a 3-plate, 96-well format; a larger version is under development. An automated 12-channel oligosynthesizer (expandable to a 96-well format) produces 12 custom oligos in 2.5 h with minimal reagent use at a significantly lower cost than commercial systems.

The group also has a semiautomated system for running gels and is developing instrumentation to simplify plasmid preparation. Other instrumentation includes a colony picker reconfigured to pick smaller libraries and a robotic library-management system. Current modules are being integrated into second-generation systems.

Informatics.

The directed strategy also has the advantage of facilitating automated data handling. The LBL informatics group, headed by Frank Eeckman, aims to collect and assemble all information from different work modules without human intervention. To accomplish this goal, the group has developed a series of computer programs that will speed construction of high-resolution physical maps, move files among different systems, and provide a visual interface for template selection. Software is being developed to support manipulation and analysis of long sequence tracts. The database Syndb is replacing 21Bdb as the primary mechanism for sharing physical-mapping and sequencing data, and annotated sequences are being submitted to GSDB.

Template-preparation and function studies.

Edward Rubin and Jan-Fang Cheng head the human biology group, which develops DNA-sequencing templates from human regions selected for their probability of containing medically important genes. A P1 contig covering 1.2 Mb of human 5q31 and containing the entire interleukin gene cluster is now the target of directed sequencing, and genetically engineered mice are being used to study the function of sequenced regions. Coupling sequence analysis with biological studies can dramatically increase knowledge of the biological and medical significance of chromosomal regions.

For more information, see http://www-hgc.lbl.gov/GenomeHome.html or contact Narla (510/486-7029, Fax: -6746).


HGMIS staff

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Human Genome Project 1990–2003

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.

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