ARS | Publication request: 454-pyrosequencing: A tool for discovery and biomarker development

Submitted to: Society of Environmental Toxicology and Chemistry Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: June 12, 2010
Publication Date: November 7, 2010
Citation: Amberg, J., Riche, M.A., Leet, J., Brown, P.B., Sepulveda, M.S. 2010. 454-pyrosequencing: A tool for discovery and biomarker development [abstract]. Society of Environmental Toxicology and Chemistry Abstracts. p. 36.

Technical Abstract: The Roche GS-FLX (454) sequencer has made possible what was thought impossible just a few years ago: sequence >1 million high-quality nucleotide reads (mean 400 bp) in less than 12 h. This technology provides valuable species-specific sequence information, and is a valuable tool to discover and understand complex physiological processes/interactions, particularly when paired with qPCR. We used these techniques to test three hypotheses: 1) gender can be determined in shovelnose sturgeon, Scaphirhynchus platorynchus, by identifying and quantifying a group of genes important in sex differentiation; 2) yellow perch, Perca flavescens, fed a diet high in soybean protein concentrate will have decreased liver size and elevated stress biomarker expression compared to fish fed a fish meal diet; and 3) decreased survival of Florida pompano, Trachinotus carolinus, maintained under low salinity conditions is caused by differential expression of solute carriers involved in osmoregulation in gills and kidneys. We produced nearly 100,000 sequences in two libraries, ovary and testis, in our first study. Unique contigs were found in the ovarian library but none in the testis library. A higher percentage of sequences that coded for genes involved in the Wnt cascade and sex differentiation (e.g. sox9, dmrt1 and spata6) were detected from ovaries. Expression of a representative number of genes from each study was verified and quantified using qPCR. Data on studies 2 and 3 will also be presented.