Robert B. Weiss, Mark Stump, Joshua Cherry, Brett Duval, Robert Black, Sandy Kazuko, Jane Macfarlane, Diane Dunn, and Peter Cartwright.
Department of Human Genetics, University of Utah, Salt Lake City, UT 84112.
We have initiated large-scale genomic DNA sequencing of microorganisms of industrial and biological interest. Our first project is to complete the genome sequence of Pyrococcus furiosus (DSM 3638), a hyperthermophilic member of the Archaea. This organism, isolated from hot marine sediments, grows vigorously at temperatures near 100°C. Novel methods and instrumentation, developed at the Utah Center for Human Genome Research, are being used [1] to sequence this ~2.0 Mb genome. The contiguous sequences of individual plasmid inserts are determined by a multiplexed transposon-based directed strategy. The University of Utah Probe Chambers, automated devices for detecting enzyme-linked fluorescence from DNA hybrids on nylon membranes, are being used in both the mapping and sequencing phase of the project.
P. furiosus plasmid and cosmid libraries have been built in a family of 21 multiplex vectors. These vectors provide multiplex tags for both the end sequencing and transposon mapping phases of the process. Common growth and DNA prep formats feed both the mapping and sequencing process. Minimal spanning sets of clones are predicted from the mapping phase and fed to the sequencing process, where cycle sequencing is performed on multiplexed doublestranded templates. Multiplex sequence ladders are transferred to nylon membranes using a direct transfer apparatus, and then placed in the Probe Chambers for multiple cycles of data collection.
Currently, over 1.5 Mb of genomic DNA clones spread across 153 plasmid and cosmid inserts have entered, and are exiting, the mapping phase. The first bolus of mapped clones that exited the mapping phase comprised 2,518 mapped priming sites spanning 0.7 Mb of genomic clones. These sets of mapped priming sites are being rapidly sequenced using two Probe Chambers, each emitted 108 sequence reads every 10.5 hr.. This initial random coverage of the genome will be superceded in the closure phase by sequence matching between newly completed insert sequences and a database of end sequences from the plasmid and cosmid libraries. This directed selection of new inserts to be sequenced, will lead to completion of the genome if all the initial gaps are sequence gaps. The Pyrococcus project will enter the closure phase in the early 1996. This project is an alpha test of methods, instrumentation and software under development at the Utah Center for Human Genome Research.
This work is funded by DOE grant DE-FG03-94ER-61950 (R.B. Weiss, P.I.)
[1] Cherry, J.L., Young, H., Di Sera, L.J., Ferguson, F.M., Kimball, A.W., Dunn, D.M., Gesteland, R.F., and Weiss, R.B. (1994). Enzyme-linked fluorescent detection for automated multiplex DNA sequencing. Genomics 20, 68-74