Expression profiling.

Coordinate gene expression during high and low shock treatments of 1 to 3 hours.

The interpretation of gene expression data requires knowledge of the diversity and relative numbers of microbial taxa. PCR products from ribosomal RNA (rRNA) coding regions reveals new levels of largely unexplored microbial diversity not represented in laboratory cultures. However, this low throughput methodology is expensive (typically $1-2/sequenced template) and sometimes inefficient when a limited number of organisms dominate a microbial population. Deoxyribonucleic acid (DNA) fingerprinting techniques such as denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) offer higher throughput but little or no taxonomic information, and many fingerprinting studies ultimately rely on DNA sequencing to characterize phylotypes.

We have developed a new, high-throughput method Serial Analysis of Gene Tags (SAGT) that provides estimates of relative frequencies and sufficient phylogenetic information to identify the phylotype of most taxa in an environmental sample. SAGT ligates together the PCR products from orthologous hypervariable regions in rRNA genes to form large concatemers. A single DNA sequencing reaction of a cloned concatemer can include as many as 20-30 orthologous hypervariable regions represented in a population of nucleic acid molecules. Samples loaded onto a 96-channel capillary sequencing machine can provide information about thousands of microorganisms in a sample. Comparison against a comprehensive rRNA gene database identifies the taxonomic assignment of individual amplicons in the concatemers. SAGT analyses of bacterial community composition in hydrothermal marine sediments from Guaymas Basin (Gulf of California) are comparable to results of cloning and sequencing of single, full-length PCR products from ribosomal RNA genes of the same microbial community. Both methods identify the same major bacterial groups. The technology allows deep sampling of microbial diversity and relative abundance in any environment.