Annotation of Sarcocystisneurona 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

BLAST DB Start End Similarity E-value Subject

BLASTX nr 41446 42924 71 2.00E-16 Conserved hypothetical protein Toxoplasma gondii

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 1.00E-37 Conserved hypothetical protein Cryptosporidium muris

BLASTX nr " " 43 1.00E-22Conserved hypothetical protein Cryptosporidium

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)

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 andToxoplasma

13

Gene ComparisonMatch of region with a conserved gene in Neospora caninum and Toxoplasmagondii

Neighbouring genes are not present in the scaffold. 14

Results – Uniprot SearchPerformed 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