Shivashankar H. Nagaraj and Shoba Ranganathan Professor and Chair – Bioinformatics Biotechnology Research Institute a

1. In silico analysis of expressed sequence tags (EST) from Trichostrongylus vitrinus (Nematoda): comparison of the automated ESTExplorer workflow platform with database searches. Shivashankar H. Nagaraj and Shoba Ranganathan Professor and Chair – Bioinformatics Biotechnology Research Institute and Adjunct Professor Dept. of Chemistry & Biomolecular Sciences Dept. of Biochemistry Macquarie University National University of Singapore Sydney, Australia Singapore (shoba.ranganathan@mq.edu.au) (shoba@bic.nus.edu.sg)

2. Expressed Sequence Tags (ESTs) • Unedited, short, single pass sequences generated from 5' or 3' end of randomly selected cDNA libraries in desired cells/tissues/organ. • Length: 200-700 bp (average 360 bp) • Can be quickly generated at low cost (“poor-man’s genome”) • EST data is highly fragmented • EST annotations have very little biological information • High-throughput in nature

3. EST Applications • Gene Discovery • Gene Structure Prediction • Expression Maps • Alternative Splicing • Identification and characterization of SNPs • Gene expression studies • tissue or disease specific • developmental stage • Proteomics (for example peptide mass fingerprinting) • Identification of drug and vaccine candidates

4. Properties of ESTs Genomic DNA mRNA cDNA ESTs An EST sequence 5’ ESTs 3’ ESTs vector repeats high quality sequence vector ~ 50 - 500 bp ~ 500- 700 bp ~ 1-50 bp High quality

5. EST data resources • Available in plenty • Several dedicated databases • Fragmented • Quality dubious • Need cleaning • Clustering • Annotation!

6. EST data repositories dbEST release 061507 (June, 2007) www.ncbi.nlm.nih.gov/dbEST/ 43,396,096 ESTs from 659 different organisms Homo sapiens (human) 8,119,106 Mus musculus (mouse) 4,850,243 Danio rerio (zebrafish) 1,350,105 Bos taurus (cattle) 1,318,208 Arabidopsis thaliana (thale cress) 1,276,692 Xenopus tropicalis 1,271,375 Oryza sativa (rice) 1,211,418 Zea mays (maize) 1,161,241 Triticum aestivum (wheat) 1,050,267

8. Overview of EST sequence analysis Submit Data Contamination check Visualize results Raw EST sequence data Vector clipping Gene annotation RNAi Gene mapping Alternative splicing SNPs Poly-A removal Repeat Masking Clustering Assembly Peptide annotation Protein interactors Gene Ontologies KEGG Consensus generation Conceptual translation

9. Evolution of ESTExplorer

10. Comparison of current methods for EST analysis Critical evaluation of contemporary tools and EST analysis pipelines Benchmarking of tools using EST datasets Lack of downstream functional annotation at DNA and protein levels ESTExplorer

11. Description of ESTExplorer

12. ESTExplorer – features • Suite of programs to pre-process, assemble and functionally annotate ESTs • User-defined input and analysis – parameter control • Species-specific analysis • Input: ESTs or assembled contigs • Output: Assembled ESTs, Gene Ontologies, mapping to Domains/Motifs, Pathway mapping

13. Phase I (EST pre-processing) Short sequences removed from the analysis Input Option 1 EST sequences SeqClean RepeatMasker Quality values (.qual) CAP3 Workflow Input Option 2 assembled ESTs Assembled ESTs Phase II (DNA level Annotation) Phase III (Protein level Annotation) ESTScan BLASTX InterProScan KOBAS BLAST2GO Final output: Annotation summary for assembled ESTs ESTExplorer analysis and annotation workflow, showing Phase I (pre-processing and assembly), Phase II (nucleotide-level annotation) and Phase III (protein-level annotation).

14. estexplorer.biolinfo.org

15. Annotation summary page

16. The worm in question • Trichostrongylus vitrinus (order Strongylida) is a parasitic nematode. • Principal causative nematode associated with parasitic diseases in sheep and cattle • Current treatment for the disease : chemotherapeutic agents (anti-helmintics) • Disadvantages with current treatments: a. Expensive and only partially effective b. Anthelminticsdrug resistance over the last decade c. Residue problems in meat and milk • Possible alternative: the development of anti-parasite drugs and/or vaccines Nisbet AJ, et al. Int J Parasitol, 2004

17. Creation of cDNA libraries and EST generation from the parasite Trichostrongylus vitrinus Bioinformatics Analysis of the ESTs Phase I Comparative genomics with nematodes Categorization of Differentially expressed ESTs Subset of potential drug target genes Phase II • Isolation of full length genes • Functional Genomics via RNAi • Biochemical activity assays • Proteomics Phase III Virtual and High-throughput screening Pre-clinical and clinical evaluation Phase IV

18. EST analysis schema

19. EST analysis schema

20. Results of overall EST analysis Number of ESTs analysed : 1776 ( male : 910 female : 866) Caenorhabditis elegans homologues 290 (41%) Homologues in parasitic nematodes 329 (42%) Homologues in non-nematodes 202 (28%) No significant match to any sequence 218 (31%) in the current databases Gene Ontologies (GO) assigned 267 (38%) Pathway associations established 230 (33%) Of the C. elegans homologues, 90 entries had observed ‘non-wildtype’ RNAi phenotypes, including embryonic lethality, maternal sterility, sterile progeny, larval arrest and slow growth.

21. Results from BLAST vs. ESTExplorer Trichostrongylus vitrinus (Nematoda: Strongylida): Molecular characterization and transcriptional analysis of Tv-stp-1, a serine/threonine phosphatase gene. Hu M, Abs El-Osta YG, Campbell BE, Boag PR, Nisbet AJ, Beveridge I, Gasser RB. Exp Parasitol. 2007 Mar 24;

22. Results from BLAST vs. ESTExplorer Trichostrongylus vitrinus (Nematoda: Strongylida): Molecular characterization and transcriptional analysis of Tv-stp-1, a serine/threonine phosphatase gene. Hu M, Abs El-Osta YG, Campbell BE, Boag PR, Nisbet AJ, Beveridge I, Gasser RB. Exp Parasitol. 2007 Mar 24;

23. Redefining parameters for possible drug/ vaccine targets in parasitic nematodes Secreted Proteins • Parasites must secrete biologically active mediators to manipulate the host environment in order to survive immune attack • Inhibit host antigen-processing pathways Examples : • Aspartyl protease inhibitor (API-1) • Cystatin (cysteine protease inhibitor) • Acetylcholinesterase (AChE) Absence of homologues in mammalian host (nematode specific genes) Strong RNAi phenotypes in C. elegans • Embryonic lethality • Larval lethality • Sterile progeny • Larval arrest • Maternal sterility • Slow growth • Genes with specificity to nematodes may serve as excellent targets for drugs/vaccines with low toxicity to humans and other vertebrates. • Better understanding of the unusual nematode biochemistry can also have industrial or therapeutic value. Harcus YM, et al. Genome Biol, 2004 Delaney A, et al. Int J Parasitol 2005 Vanholme B, et al. Gene 2004

24. T. vitrinus male EST data comparison C. elegans Venn diagram 169 (39.21%) 19 6 55 89 3 45 2 191 (44.31%) 100 (23.20%) Parasitic nematodes Non-nematodes T. vitrinus female EST data comparison C. elegans Venn diagram 121 (45.6%) 6 24 6 85 3 8 26 Parasitic nematodes Non-nematodes 102 (38.4%) 138 (52.1%)

25. SimiTri : visualizing similarity relationships for groups of sequences Database 1 BLAST Query dataset (EST sequences in this study) SimiTri provides a two-dimensional display of relative similarity relationships among three different datasets. Database 2 Database 3 vizualization • Java/Perl-based application • Display of relative similarity relationships • Analysis of relative similarity relationships • Based on raw bit score from BLAST output Parkinson J, et al. Bioinformatics, 2003 Parkinson J, et al. Nat Genetics, 2004

26. Color scale of maximal BLAST scores for tiles a. SimiTri: Male dataset SimiTri results: T. vitrinus ESTs are closer to parasitic nematodes and C. elegans than to other non-nematode organisms. C. elegans 431 male ESTs 19 169 (39.21%) 100 150 200 250 300 No match for 114 ESTs 6 55 100 89 3 45 2 Parasitic nematodes Non-nematodes 191 (44.31%) 100 (23.20%) Color scale of maximal BLAST scores for tiles b. SimiTri : Female dataset C. elegans 6 121 (45.6%) 265 female ESTs 100 150 200 250 300 No match for 78 ESTs 6 24 100 85 8 26 3 Parasitic nematodes Non-nematodes 102 (38.4%) 138 (52.1%)

27. BLAST vs. ESTExplorer • ESTExplorer reliably and rapidly annotated 301 ESTs, with pathway and GO information, eliminating 60 low quality hits from database searches. 1776 ESTs 1776 ESTs Analysis using semi-automated approach via ESTExplorer Analysis of individual ESTs using BLAST • Slow (took several weeks) • BLAST results are the only evidence for functional assignment • Peripheral annotation • Fast (took few minutes) • Multiple evidences for annotation supported by GO, InterProScan and Pathway Mapping • In depth annotation

28. Secreted protein analysis Number of putative secreted proteins : 40 Immune-response related genes Signalling molecules Ion channels Proteases Protease inhibitors

29. Candidate target genes in Trichostrongylus vitrinus

30. Results from BLAST vs. ESTExplorer Trichostrongylus vitrinus (Nematoda: Strongylida): Molecular characterization and transcriptional analysis of Tv-stp-1, a serine/threonine phosphatase gene. Hu M, Abs El-Osta YG, Campbell BE, Boag PR, Nisbet AJ, Beveridge I, Gasser RB. Exp Parasitol. 2007 Mar 24;

31. Results from BLAST vs. ESTExplorer Trichostrongylus vitrinus (Nematoda: Strongylida): Molecular characterization and transcriptional analysis of Tv-stp-1, a serine/threonine phosphatase gene. Hu M, Abs El-Osta YG, Campbell BE, Boag PR, Nisbet AJ, Beveridge I, Gasser RB. Exp Parasitol. 2007 Mar 24;

32. ESTExplorer : applications so far .. 1. In silico analysis of expressed sequence tags (EST) from Trichostrongylus vitrinus (Nematoda): comparison of the automated ESTExplorer workflow platform with database searches.Nagaraj SH, Gasser RB, Ranganathan S. 2. A transcriptomic analysis of the adult stage of the bovine lungworm, Dictyocaulus viviparus. Ranganathan S, Nagaraj SH, Hu M, Strube C, Schnieder T and Gasser RB. BMC Genomics, 2007, accepted 3. Gender-enriched transcripts in adult Haemonchus contortus (Nematoda) – predicted functions and genetic interactions based on comparative analyses with Caenorhabditis elegans. Campbell BE, Nagaraj SH, Hu M, Zhong W, Sternberg PW, Ong EK, Loukas A, Ranganathan S, Beveridge A and Robin B. Gasser. 4. Transcriptional changes in the third-stage larva of Ancylostoma caninum (Nematoda) following in vitro serumstimulation, employing a suppressive-subtractive hybridisation-based microarray approach. Datu BJD, Gasser RB, Nagaraj SH, Eng K. Onge, O’Donoghue P, McInnes R, Ranganathan S and Loukas A 5. Trichostrongylus vitrinus (Nematoda: Strongylida): Molecular characterization and transcriptional analysis of Tv-stp-1, a serine/threonine phosphatase gene. Hu M, Abs El-Osta YG, Campbell BE, Boag PR, Nisbet AJ, Beveridge I, Gasser RB. Exp Parasitol. 2007, accepted

33. Ref papers

34. Acknowledgements Prof. Robin Gasser (University of Melbourne) Genetics Technologies Pty. Ltd. Australian Research Council LINKAGE PROJECT (LP0667795)

36. Some more examples of secreted proteins M41 family metalloproteasemitochondrial membrane proteinase : Schistosoma Pathogenesis related protein similar to helminth venom allergen homologues :Schistosoma