Annotation of Sarcocystis neurona scaffolds Nigel Austin Turgay

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 DB Start End Similarity E-value Subject BLASTX nr 41446 42924 71 2. 00 E-16 BLASTX nr 41464 42942 42 2. 00 E-44 Conserved hypothetical protein Plasmodium falciparum BLASTX nr " " 44 2. 00 E-42 Conserved hypothetical protein Plasmodium vivax BLASTX nr " " 41 1. 00 E-37 Conserved hypothetical protein Plasmodium berghei BLASTX nr " " 40 BLASTX nr " " 43 1. 00 E-37 Conserved hypothetical protein Cryptosporidium muris Conserved hypothetical protein Cryptosporidium 1. 00 E-22 parvum BLASTX nr 10632 10992 69 6. 00 E-33 Putative lectin doman protein Toxoplasma gondii BLASTX nr 32690 32968 66 7. 00 E-18 Transcript GF 18541 Drosophila melanogaster BLASTX nr " " 66 6. 00 E-17 Putative acylphosphatase Aedes aegypti BLASTX nr " " 69 4. 00 E-16 Putative acylphosphatase Toxoplasma gondii TBLAST est 1538 1840 45 5. 00 E-08 Xenopus m. RNA (c. DNA library) TBLAST est " " 51 1. 00 E-07 Cyprinus carpio m. RNA (c. DNA library) TBLAST est 10986 10967 82 5. 00 E-10 T. gondii m. RNA (c. DNA library) TBLAST est 14716 14904 87 2. 00 E-33 T. gondii m. RNA (c. DNA 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 - Gene 1 • 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 GENE 1 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 (Gene 1) was found in 400 Kb of DNA – scaffolds perhaps in a gene poor region of genome • Gene 1 is perhaps orthologous with a gene in Toxoplasma and Neurospora • Hypothetical gene – no function prescribed to it 19

Thank You!!! 20