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Introduction to the Workshop
URLs Provided by Attendees

Abstracts

Mapping

Informatics

Sequencing

Instrumentation

Ethical, Legal, and Social Issues

Infrastructure

The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.

Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.

Automation of a Directed Sequencing Strategy

Joseph M. Jaklevic
Human Genome Center and Engineering Division, Lawrence Berkeley Laboratory, University of California, Berkeley, CA 94720

The Lawrence Berkeley Laboratory Human Genome Center is currently expanding a large-scale directed sequencing project toward a multi-megabase per year capability. The LBL directed sequencing strategy is based on STS content mapping of P-1 inserts followed by high-resolution mapping of 3 kb fragments and transposon assisted sequencing. This approach results in a tenfold reduction in template preparation and a five-fold reduction in sequencing relative to random strategies. Automation of this directed approach presents many unique challenges and opportunities in the area of laboratory instrumentation. In particular, the extensive high-resolution mapping which precedes template sequencing places large demands upon PCR gel assays and related protocols.

Our approach to automation of this strategy includes the use of specific robotics-based tools for colony picking, library replication, pooling and for performing PCR reaction preps and sequencing reactions. In addition, a number of specialized instrumentation modules have been developed. An automated image acquisition and analysis system employs a digital camera to acquire images from ethidium bromide stained gels. Image processing and analysis software is then used to automatically locate bands and assign sizes for mapping purposes. A robotics-compatible, multi-station thermal cycler is currently capable of performing 600 amplifications per hour in a three-plate, 96-well format. The next version will have a larger number of individual stations and will be capable of using 384-well plates. An automated 12-channel oligosynthesizer produces custom oligos at a rate and cost significantly improved with respect to existing commercial systems. It is designed to be expandable in multiples of 12 channels up to a 96-well format. Progress in all of the areas will be reviewed.

Recent efforts to integrate these specialized modules into functional units with robotic materials handling and data tracking will also be presented.