Transcriptome analysis by pyrosequencing: A new tool for assessing the response of marine animals to environmental stress

Sibel I. Karchner, Biology
Mark E. Hahn, Biology

Grant Funded 2008

Marine organisms are continually challenged by changing environmental conditions, including wide variations in temperature, pressure, salinity, oxygen tension, light, nutrient availability, or the concentrations of toxic chemicals (natural or anthropogenic). They respond to this variable environment in large part through changes in gene expression, which underlie adaptive physiological capabilities that serve to mitigate the environmental stress. Study of these adaptive changes in terrestrial and laboratory model organisms has been facilitated by the increasing number of sequenced genomes of these organisms, and the associated microarray resources for performing gene expression profiling. However, for the vast majority of marine organisms—and marine animals in particular—such resources are lacking. For non-model organisms without sequenced genomes, current approaches for measuring altered gene expression on a genome-scale are inadequate or prohibitively expensive.

Recently, the “next generation” of DNA sequencing technologies has increased the throughput and reduced the cost of DNA sequencing by orders of magnitude. The method of pyrosequencing on high-density picoliter plates pioneered by 454 Life Sciences (now Roche Applied Sciences) combines high-throughput (400,000 individual sequences per run) with long reads (currently ~250 bp, soon to be 400 bp) and high accuracy. We propose to conduct experiments that will provide proof of concept regarding the application of 454 pyrosequencing to studies of gene expression in response to environmental stress in marine metazoans. To do this, we will characterize the transcriptome of the Atlantic killifish (mummichog) (Fundulus heteroclitus) and measure the transcriptional response to environmental stresses, using hypoxia and toxic chemicals as examples. These studies will be the first application of pyrosequencing technology to combine transcriptome characterization and gene expression profiling in a marine metazoan. The pyrosequencing approach has great potential for application to a variety of problems in marine science, including studies in ecology, evolution, population biology, life history transitions, and adaptation to extreme and variable environments.