Practice of slide show

1. A new procedure for determining the genetic basis of a physiological process in a non-model species, illustrated by cold induced angiogenesis in the carp PhD dinner seminars John Herbert

2. Why angiogenesis due to cold temperature? It is more difficult for muscles to utilise O 2 at lower temperatures

3. What Evidence? Cold induced angiogenesis has been shown in mammals and vertebrates 25 o C 5 o C

4. In the non-model species carp (no genome and little transcriptome ~20K EST sequences), ortholog relationships and microarray re-analyses were used to predict identity of cold induced angiogenic genes

5. Functionally linked together by orthologs Basic strategy overview What is an ortholog and how to create this link with 20K anonymous carp DNA sequences Model species Angiogenic genes Non-model species Cold induced genes

6. Bioinformatic Strategy 1) Ortholog prediction 3) Cold induced genes 2) Angiogenic genes 20K ESTs 17K Contigs Cold induced angiogenesis genes

7. How to find orthologs? Kuzniar A, van Ham RC, Pongor S, Leunissen JA: The quest for orthologs: finding the corresponding gene across genomes . Trends Genet 2008, 24 (11):539-551.

8. BLAST on orthologous groups

9. BLASTO illustrates limitations with public available software Zhou Y, Landweber LF: BLASTO: a tool for searching orthologous groups . Nucleic Acids Res 2007, 35 (Web Server issue):W678-682 Database from 2007 Singular, just one sequence Limited filtering options

11. Reciprocal Best Hit (RBH): Ortholog assignment using BLAST Human Carp

13. Choice of BLAST filter of low complexity makes a difference

15. Eukaryotic Filter comparison

16. How to improve on this? Zebrafish Another model species Genome is almost complete Currently 6 million ESTs 28 thousand Refseq proteins Carp……….only…. 20K ESTs Diverged 50 MYA Use zebrafish as stepping stone (SRBH)

17. Genome duplication

18. Teleost genome duplication: can be seen with genes important in endothelial biology Human chromosome 11 Zebrafish chromosome 18 (on other strand) Zebrafish chromosome 16

19. Stepped Reciprocal Best Hit Zebrafish genome ~complete: 28K Proteins; 6Million ESTs

20. Seq: AU052068 versus zebrafish and human SRBH: This was not perfect

21. Conditional Stepped Reciprocal Best Hit

22. CSRBH method of choice

23. CSRBH 52% successful SRBH 49% RBH 41%

24. Gracey et al. 2004: Coping with cold: An integrative, multitissue analysis of the transcriptome of a poikilothermic vertebrate. 17,825 sequences on chip This data set proved appropriate for meta-analysis to predict cold-induced angiogenic genes in skeletal and heart muscle tissues. Cold induced genes

25. Carp gene expression cooling regimen B) 57 genes up-regulated on initial cooling 567 total non-redundant genes up-regulated A) 547 persistently up-regulated genes

27. Combining results found 135 cold induced angiogenesis genes in carp Angiogenic gene prediction 908 Cold induced genes 567 Ortholog predictions 3762 135

28. 12 genes picked out from 135 up-reg. angiogenesis genes in response to cold

32. Good news, BMC genomics accepted it just on Friday 