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.

Microbial Genome Sequencing

Douglas R. Smith and John Reeve[1]
Genome Therapeutics Corporation
[1]Ohio State University

The goal of this project is to determine the sequence of several microbial genomes of particular importance from the perspectives of energy production and bioremediation. The target organisms are Methanobacterium thermoautotrophicum (1.7 Mb), chloroplast (100 kb), Rhodococcus rhodochrous (2.5 Mb), Methanopyrus kandleri (1.7 Mb), Synechococcus sp. (2.7 Mb), and Haloferax volcanii (4.2 Mb). The sequencing will be done using state-of-the-art multiplex sequencing technology and will build on our current success in generating and analyzing around 1 Mb of finished and fully annotated genomic sequence from the Mycobacterium leprae and M. tuberculosis genomes.

Each genome will be shotgun sequenced in its entirety at about eightfold random coverage. Clone pooling, DNA preparation, and cycled dideoxy sequencing reactions will be performed using established procedures and robotic instrumentation wherever applicable. Finish sequencing will be performed by cycled sequencing methods from subclone pools. Data acquisition will be accomplished by digital film or infrared fluorescence scanning. Sequences will be read on computer workstations using an automated image-analysis program, REPLICA(tm) [developed by L. Mintz and G. Church (HHMI at Harvard Medical School)]. The sequences will be assembled into contigs, and the contigs will be proofread using a modified GelAssemble platform and REPLICA. Analysis for genes and structural features will be carried out with a variety of programs including a specialized set we are currently using for the analysis of large bacterial genomic regions (Large Sequence Analysis Suite).

Mathematical modeling of shotgun sequencing was done using the mathematically rigorous equations of Lander and Waterman (1988) assuming a read length of 400 bases and an 80% overall success rate. These calculations predict that, at eightfold coverage, a 1.7-Mb genome is expected to assemble into 19 contigs averaging 90 kb in size. The gaps between contigs are predicted to be small (2.3 x 10(-4) chance that a gap will be greater than 400 bp) and a bridgeable (1.6 x 10(-9) chance that a gap could not be bridged by walking on one of the existing 2-kb shotgun sequencing templates).

All sequences will be made available with full annotation for gene locations as soon as possible (and certainly within 6 months) after completion. Sequences will be submitted using automated ASN1 routines, which we are currently using to annotate mycobacterial cosmid sequences. To facilitate biochemical studies, the Ohio State group will generate and distribute a resource of sequence-mapped clones (lambda or cosmid) for each genome.

This work is funded by a cooperative agreement between DOE and Genome Therapeutics Corp. (DE-FC02-95ER61967).