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Annotation of Sarcocystis neurona scaffolds Nigel Austin Turgay Ibrikci Liliana Lopez Kleine Marton Megyeri Caribbean Training Programme on Bioinformatics January 2010
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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
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S. neurona & Related Apicomplexa 3 Sarcocystis neurona Eimeria Neospora Toxoplasma
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Life Cycle of S. neurona 4
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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
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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
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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
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Results – Blast Search 8
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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)
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ACT Results 10 Match of region with a conserved gene in Neospora caninum and Toxoplasma gondii Neospora caninum scaffolds
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Hmmm…. No genes in 400,000 bp DNA??? And then…. Expertise, experience He was able to locate a gene 11
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Gene Discovered! Match of region with a conserved gene in Neospora caninum 12
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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
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Gene Comparison Match of region with a conserved gene in Neospora caninum and Toxoplasma gondii Neighbouring genes are not present in the scaffold. 14
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Results – Uniprot Search Performed with GENE1 15
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Further Protein Info Characterize our protein product – Membrane protein? High regions of hydrophobicity – Domains and motifs – Secondary structures 16
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No transmembrane motifs present Hydrophobicity Graph 17
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Domains & Motifs 18
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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
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Thank You!!! 20
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