DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

124. Pangenomic Microbial Comparisons by Subtractive Hybridization

Peter Agron, Lyndsay Radnedge, Evan Skowronski, Madison Macht, Jessica Wollard, Sylvia Chin, Aubree Hubbell, Marilyn Seymour, Christina Nocerino, and Gary Andersen

Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, CA 94550

andersen2@llnl.gov

Sequencing of whole genomes is reshaping microbiology. However, as more sequence information is generated, there will be increased sequence redundancy between closely related species or strains. In the course of time, the amount of new sequence information obtained by whole genome sequencing with current technology will become increasingly less cost efficient. We are exploring the use of suppression subtractive hybridization (SSH) of total DNA as a means of focusing sequencing efforts on unique regions when a reference strain of known sequence is compared to a different isolate of the same species or genus. To rigorously examine this approach, two sequenced strains of Helicobacter pylori (J99 and 26695) were used as a model system, as this allows rapid determination and mapping of difference products based on sequencing alone. Using the high-throughput SSH methods, difference products can be rapidly cloned, sequenced, and then mapped by comparing the data to the H. pylori genome database. To increase the likelihood of amplifying difference products from any given region, several restriction enzymes were used in separate SSH experiments. So far we have obtained data from 2,123 clones that reveal 427 (20%) unique sequences. Control subtractions with an Escherichia coli strain containing the transposon Tn5 against its isogenic parent showed a 270-fold enrichment for Tn5 sequences, demonstrating that SSH is highly effective. Current efforts are focused on: 1) mapping of the difference products onto the relevant genome using the cross_match algorithm and Percent Identity Plots, 2) assessing coverage of the difference regions by the subtracted clones, 3) assessing the redundancy of this coverage and 4) determining the reproducibility of SSH. We will present data that address the overall efficacy of the use of subtractive hybridization for pangenomic microbial comparisons.