Annotation Presentation Week 2

1. Annotation Presentation Week 2 Pathway Assignments

2. The assignment – Annotating Pathways KEGG Pathway Database http://www.genome.jp/kegg/pathway.html

3. Metabolism Energy Nitrogen metabolism The assignment – Annotating Pathways KEGG Pathway Database All teams will be assigned a different pathway. This one is provided only as an example.

4. Which genes are found in P. limnophilus? Closely related to P. limnophilus The green boxes correspond to genes found in R. baltica Hypothesis: If P. limnophilus uses nitrogen as NRG source, then it likely will have the same genes as R. baltica.

5. EC Numbers Search by EC number http://www.expasy.org/enzyme/

6. EC Numbers

7. Biochemical Pathways Field M10 of the Roche Applied Science “Biochemical Pathways” wall chart

8. EC Numbers

9. MEDLINE Literature

10. Other Scholarly Literature

11. What do you do with assigned gene? 1- Go to KEGG Pathway Database 2- Click on “green box” corresponding to the gene that was assigned to you. “Click”

12. KEGG Information 3- Obtain amino acid sequence (FASTA format) from KEGG for assigned R. baltica gene “Click” for FASTA file Amino acid sequence

13. KEGG Information 4- “COPY/PASTE” entire entry (including description line) into text editing program (i.e., Notepad, Wordpad) for use in subsequent database searches • FASTA format: • Description line preceded by greater than sign • Amino acid sequence for protein encoded by corresponding gene • NOTE: Nucleotide sequences also can be depicted in FASTA format

14. Then what? 5- In the text editor (Notepad), save the file containing the amino acid sequence in FASTA format as plain text file (.txt) 6- Return to imgACT

15. Reciprocal BLAST search 9- Right click on img/edu icon and open Organism Details page for Planctomyces limnophilus DSM 3776 Get used to right-click then open in new tab “Click” “Click” 10- Click “Find Genes” tab

16. Reciprocal BLAST search 11- Click [BLAST] from menu in “Find Genes” tab “Click” 12- Retrieve amino acid sequence for R. baltica gene. Copy/paste into query box. Select blastp program with E-value cutoff of 1e-2 and P. limnophilus genome as the database to be searched. Then hit <Run BLAST>.

17. Reciprocal BLAST search Referred to as a“Reciprocal BLAST” search in instructions

18. Reciprocal BLAST search 13- Results of BLAST search will be displayed in format similar to what is generated with NCBI database Inspect “hit” list

19. Reciprocal BLAST search 14- Select best “hit” and add to “Gene Cart” Scroll down

20. Reciprocal BLAST search Note Gene Object ID (OID) 14- Return to imgACT

21. Getting Started with IMG-ACT • imgACT “Student Teams” will be created by the instructor once all students have registered for course • - Find your course “Home” page on imgACT Select MIMG 103L, Winter 2010

22. Getting Started • View your team(s) by clicking on Student Team name “Click” “Click” Josie Bruin josie@ucla.edu Joe Bruin joe@ucla.edu Jenny Bruin jenny@ucla.edu Jess Bruin jess@ucla.edu Jeffrey Bruin jeff@ucla.edu

23. Getting Started • View your Practice Gene by clicking link under “Gene Annotation Assignments” “Click”

24. Setting up lab notebook for annotation assignment • View your team ASSIGNMENT by clicking link under “Pathway/Structure Assignments” “Click”

25. Setting up lab notebook for annotation assignment You should see the “Pathway/Structure Report” page Notice team memberswith email addresses (Your TA & instructors also will be listed here)

26. Setting up lab notebook for annotation assignment As a TEAM: set up your team notebook page “Click”

27. Setting up lab notebook for annotation assignment Add pathway & gene information for reference organism Insert KEGG map: - return to KEGG database - save KEGG map as .gif or .png

28. Setting up lab notebook for annotation assignment “Click” Must be in EDIT mode to insert image “Click” Place cursor where you want to insert image “Click” NOTE: Upload entireKEGG map. Only cropthe image if assignedstructure/complex orpathway is portion of full KEGG map “Click”

29. Setting up lab notebook for annotation assignment Result after image inserted and comments added to box:

30. Setting up lab notebook for annotation assignment Each person on the team should enter the informationfor the Reference Organism & Gene used for theirReciprocal BLAST search: • This step requires that • you create additional • boxes and headings.While in EDIT mode,simply copy/paste thenmodify the text. • Include full name ofreference organism. • Include amino acidsequence used for the • reciprocal BLAST search • (copy/paste from Notepad).

31. Setting up lab notebook for annotation assignment Press SAVE button so you don’t lose your work. “CLICK” • IMPORTANT NOTES!! • Only ONE person at a time from each team may modify the team notebook; otherwise you will delete your teammate’s work! • Skip MetaCyc section – you may add this later in the quarter. NOTE: Additional information for MetaCyc will be entered on the page later.

32. Then what? Return to “Pathway/Structure Report” in imgACT Individually add steps or components to your pathway or structure “Click”

33. Then what? Enter the “Step name” and “Gene Object ID”. Then press <SAVE>. Your TA may have suggestions for “step name” (or component name for cell structures) if you are not sure what to call it. The Gene Object ID corresponds to top reciprocal BLAST hit inP. limnophilus genome.

34. Then what? “Pathway/Structure Report” page should be updated Indicates step creationwas successful

35. Then what? Log out of lab notebook

36. Then what? Sign out of imgACT “CLICK” Close all tabs and windows before exiting the browser