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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 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 Sarcocystis neurona Eimeria Neospora Toxoplasma 3 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 BLAST DB Start End Similarity E-value Subject BLASTX nr 41446 42924 71 2.00E-16 BLASTX nr 41464 42942 42 2.00E-44 Conserved hypothetical protein Plasmodium falciparum BLASTX nr " " 44 2.00E-42 Conserved hypothetical protein Plasmodium vivax BLASTX nr " " 41 1.00E-37 Conserved hypothetical protein Plasmodium berghei BLASTX nr " " 40 BLASTX nr " " 43 1.00E-37 Conserved hypothetical protein Cryptosporidium muris Conserved hypothetical protein Cryptosporidium 1.00E-22 parvum BLASTX nr 10632 10992 69 6.00E-33 Putative lectin doman protein Toxoplasma gondii BLASTX nr 32690 32968 66 7.00E-18 Transcript GF18541 Drosophila melanogaster BLASTX nr " " 66 6.00E-17 Putative acylphosphatase Aedes aegypti BLASTX nr " " 69 4.00E-16 Putative acylphosphatase Toxoplasma gondii TBLAST est 1538 1840 45 5.00E-08 Xenopus mRNA (cDNA library) TBLAST est " " 51 1.00E-07 Cyprinus carpio mRNA (cDNA library) TBLAST est 10986 10967 82 5.00E-10 T. gondii mRNA (cDNA library) TBLAST est 14716 14904 87 2.00E-33 T. gondii mRNA (cDNA library) Conserved hypothetical protein Toxoplasma gondii 9 ACT Results Match of region with a conserved gene in Neospora caninum and Toxoplasma gondii Neospora caninum scaffolds 10 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 Hydrophobicity Graph No transmembrane motifs present 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