In silico analysis of expressed sequence tags from Trichostrongylus vitrinus (Nematoda): comparison of the automated ESTExplorer workflow platform with conventional database searches.
BACKGROUND
The analysis of expressed sequence tags (EST) offers a rapid and cost effective approach to elucidate the transcriptome of an organism, but requires several computational methods for assembly and annotation. Researchers frequently analyse each step manually, which is laborious and time consuming. We have recently developed ESTExplorer, a semi-automated computational workflow system, in order to achieve the rapid analysis of EST datasets. In this study, we evaluated EST data analysis for the parasitic nematode Trichostrongylus vitrinus (order Strongylida) using ESTExplorer, compared with database matching alone.


RESULTS
We functionally annotated 1776 ESTs obtained via suppressive-subtractive hybridisation from T. vitrinus, an important parasitic trichostrongylid of small ruminants. Cluster and comparative genomic analyses of the transcripts using ESTExplorer indicated that 290 (41%) sequences had homologues in Caenorhabditis elegans, 329 (42%) in parasitic nematodes, 202 (28%) in organisms other than nematodes, and 218 (31%) had no significant match to any sequence in the current databases. Of the C. elegans homologues, 90 were associated with 'non-wildtype' double-stranded RNA interference (RNAi) phenotypes, including embryonic lethality, maternal sterility, sterile progeny, larval arrest and slow growth. We could functionally classify 267 (38%) sequences using the Gene Ontologies (GO) and establish pathway associations for 230 (33%) sequences using the Kyoto Encyclopedia of Genes and Genomes (KEGG). Further examination of this EST dataset revealed a number of signalling molecules, proteases, protease inhibitors, enzymes, ion channels and immune-related genes. In addition, we identified 40 putative secreted proteins that could represent potential candidates for developing novel anthelmintics or vaccines. We further compared the automated EST sequence annotations, using ESTExplorer, with database search results for individual T. vitrinus ESTs. ESTExplorer reliably and rapidly annotated 301 ESTs, with pathway and GO information, eliminating 60 low quality hits from database searches.


CONCLUSIONS
We evaluated the efficacy of ESTExplorer in analysing EST data, and demonstrate that computational tools can be used to accelerate the process of gene discovery in EST sequencing projects. The present study has elucidated sets of relatively conserved and potentially novel genes for biological investigation, and the annotated EST set provides further insight into the molecular biology of T. vitrinus, towards the identification of novel drug targets.
Nagaraj
Shivashankar H
SH
Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW 2109, Australia. snagaraj@els.mq.edu.au
Gasser
Robin B
RB
Nisbet
Alasdair J
AJ
Ranganathan
Shoba
S
D000818:Animals; D001483:Base Sequence; D030541:Databases, Genetic; D020224:Expressed Sequence Tags; D008969:Molecular Sequence Data; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D013045:Species Specificity; D014157:Transcription Factors / Q000235:genetics*; D014254:Trichostrongylus / Q000235:genetics*
D014157:Transcription Factors