Generation and analysis of expressed sequence tags from the ciliate protozoan parasite Ichthyophthirius multifiliis

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figure 1

Venn diagram summary of sequence comparisons of the Ich ESTs with Tetrahymena thermophila and Plasmodium falciparum genomes. A total of 4,706 unique Ich ESTs were used as queries yielding 1,759 significant (E-value < 10^-5) hits to the T. thermophila genome, and 817 to the P. falciparum genome. A total of 695 sequences were ESTs with common hits to both genomes.

Figure 2

Pie charts of 2^nd level gene ontology (GO) terms. Overall, 1,008 unique sequences were annotated using the Blast2GO software and included in the graphs. Each of the three GO categories is presented including cellular component (a), molecular function (b), and biological process (c).