Generation and analysis of expressed sequence tags from the ciliate protozoan parasite Ichthyophthirius multifiliis
1 The Fish Molecular Genetic and Biotechnology Laboratory, Department of Fisheries and Allied Aquacultures and Program of Cell and Molecular Biosciences, Aquatic Genomics Unit, Auburn University, AL 36849 USA
2 Aquatic Animal Health Research Laboratory, Agricultural Research Service, United States Department of Agriculture, Post Office Box 952, Auburn, AL 36831 USA
BMC Genomics 2007, 8:176 doi:10.1186/1471-2164-8-176Published: 18 June 2007
The ciliate protozoan Ichthyophthirius multifiliis (Ich) is an important parasite of freshwater fish that causes 'white spot disease' leading to significant losses. A genomic resource for large-scale studies of this parasite has been lacking. To study gene expression involved in Ich pathogenesis and virulence, our goal was to generate expressed sequence tags (ESTs) for the development of a powerful microarray platform for the analysis of global gene expression in this species. Here, we initiated a project to sequence and analyze over 10,000 ESTs.
We sequenced 10,368 EST clones using a normalized cDNA library made from pooled samples of the trophont, tomont, and theront life-cycle stages, and generated 9,769 sequences (94.2% success rate). Post-sequencing processing led to 8,432 high quality sequences. Clustering analysis of these ESTs allowed identification of 4,706 unique sequences containing 976 contigs and 3,730 singletons. These unique sequences represent over two million base pairs (~10% of Plasmodium falciparum genome, a phylogenetically related protozoan). BLASTX searches produced 2,518 significant (E-value < 10-5) hits and further Gene Ontology (GO) analysis annotated 1,008 of these genes. The ESTs were analyzed comparatively against the genomes of the related protozoa Tetrahymena thermophila and P. falciparum, allowing putative identification of additional genes. All the EST sequences were deposited by dbEST in GenBank (GenBank: EG957858–EG966289). Gene discovery and annotations are presented and discussed.
This set of ESTs represents a significant proportion of the Ich transcriptome, and provides a material basis for the development of microarrays useful for gene expression studies concerning Ich development, pathogenesis, and virulence.