1. Annotation of Sarcocystis neurona scaffolds Nigel Austin Turgay Ibrikci Liliana Lopez Kleine Marton Megyeri Caribbean Training Programme on Bioinformatics January 2010

2. Sarcocystis neurona Genus: Sarcocystis - parasitic protozoa occur as sporocysts in the muscle of mammals, birds, and reptiles. In humans – asymptomatic Sarcocystis neurona causes equine protozoal myoencephalitis 2

3. S. neurona & Related Apicomplexa 3 Sarcocystis neurona Eimeria Neospora Toxoplasma

4. Life Cycle of S. neurona 4

5. About Data Data cordially supplied by Dr. Jessica Kissinger who very recently acquired the genome sequence First 120,000 bp in 4 scaffolds – analysis Then 400,000 bp in 4 scaffolds - analysis 5

6. Objectives To annotate novel DNA sequences of S. neurona. Detection of coding sequences by: – comparison with other sequences in data bases NB: No reference genome or other info was available since sequences were novel 6

7. Strategy for Scaffolds BLASTX in nr db: search of translated sequence in protein databases TBLASTX in est db: search of translated sequence in translated sequence databases Comparison in ACT with most closely related organisms (Toxoplasma gondii and Neospora caninum) 7

8. Results – Blast Search 8

9. Results BLAST 9 BLASTDBStartEndSimilarityE-valueSubject BLASTXnr4144642924712.00E-16Conserved hypothetical protein Toxoplasma gondii BLASTXnr4146442942422.00E-44Conserved hypothetical protein Plasmodium falciparum BLASTXnr""442.00E-42Conserved hypothetical protein Plasmodium vivax BLASTXnr""411.00E-37Conserved hypothetical protein Plasmodium berghei BLASTXnr""401.00E-37Conserved hypothetical protein Cryptosporidium muris BLASTXnr""431.00E-22 Conserved hypothetical protein Cryptosporidium parvum BLASTXnr1063210992696.00E-33Putative lectin doman protein Toxoplasma gondii BLASTXnr3269032968667.00E-18Transcript GF18541 Drosophila melanogaster BLASTXnr""666.00E-17Putative acylphosphatase Aedes aegypti BLASTXnr""694.00E-16Putative acylphosphatase Toxoplasma gondii TBLASTest15381840455.00E-08Xenopus mRNA (cDNA library) TBLASTest""511.00E-07Cyprinus carpio mRNA (cDNA library) TBLASTest1098610967825.00E-10T. gondii mRNA (cDNA library) TBLASTest1471614904872.00E-33T. gondii mRNA (cDNA library)

10. ACT Results 10 Match of region with a conserved gene in Neospora caninum and Toxoplasma gondii Neospora caninum scaffolds

11. Hmmm…. No genes in 400,000 bp DNA??? And then…. Expertise, experience He was able to locate a gene 11

12. Gene Discovered! Match of region with a conserved gene in Neospora caninum 12

13. Discovered Gene - Gene1 The discovered gene was expanded on both the 5’ and 3’ end Start and stop codons were identified Protein sequence was determined BLAST – hypothetical protein with high similarity to one found in Neospora and Toxoplasma 13

14. Gene Comparison Match of region with a conserved gene in Neospora caninum and Toxoplasma gondii Neighbouring genes are not present in the scaffold. 14

15. Results – Uniprot Search Performed with GENE1 15

16. Further Protein Info Characterize our protein product – Membrane protein? High regions of hydrophobicity – Domains and motifs – Secondary structures 16

17. No transmembrane motifs present Hydrophobicity Graph 17

18. Domains & Motifs 18

19. Conclusion Various blast searches may assist in location of orthologous genes in other genomes ACT very useful tool for gene discovery and annotation (along with experience & expertise) One gene (Gene1) was found in 400 Kb of DNA – scaffolds perhaps in a gene poor region of genome Gene1 is perhaps orthologous with a gene in Toxoplasma and Neurospora Hypothetical gene – no function prescribed to it 19

20. Thank You!!! 20