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

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

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

Life Cycle of S. neurona 4

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

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

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

Results – Blast Search 8

Results BLAST 9 BLASTDBStartEndSimilarityE-valueSubject BLASTXnr E-16Conserved hypothetical protein Toxoplasma gondii BLASTXnr E-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 BLASTXnr E-33Putative lectin doman protein Toxoplasma gondii BLASTXnr E-18Transcript GF18541 Drosophila melanogaster BLASTXnr""666.00E-17Putative acylphosphatase Aedes aegypti BLASTXnr""694.00E-16Putative acylphosphatase Toxoplasma gondii TBLASTest E-08Xenopus mRNA (cDNA library) TBLASTest""511.00E-07Cyprinus carpio mRNA (cDNA library) TBLASTest E-10T. gondii mRNA (cDNA library) TBLASTest E-33T. gondii mRNA (cDNA library)

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

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

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

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

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

Results – Uniprot Search Performed with GENE1 15

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

No transmembrane motifs present Hydrophobicity Graph 17

Domains & Motifs 18

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

Thank You!!! 20