Introduction to Bioinformatics Wednesday, 2 March 2011 Genome analysis

1. Introduction to BioinformaticsWednesday, 2 March 2011Genome analysis • Introduction to Wally and Batiatus • Organization of research groups • Hatfull et al (2008) • Break up into research groups This demonstration is best viewed as a slide show,enabling you to simulate a session and make changes in cursor position more obvious.To do this, click Slide Show on the top tool bar, then View show. Click anywhere to go on to the next slide

2. Viral Genomes Project

3. Viral Genomes Project Wally Batiatus Rachel Walstead and Danielle Renner

4. Introduction to BioinformaticsWednesday, 2 March 2011Genome analysis • Introduction to Wally and Batiatus • Organization of research groups • Hatfull et al (2008) • Break up into research groups

5. Organization of Project The organization of the project can be found on the Genome Analysis module page

6. Organization of Project The page has the same sort of things that you’ve become used to in module pages – a place to find notes, problem sets, etc But in addition to all that (scroll down)…

7. Organization of Project • …there’s a section on analysis groups. • It’s divided into two parts: • Annotation of our two phage, Wally and Batiatus • Analysis of different aspects of bacteriophage genomes • First, phage annotation. Let me give an example, using BioBIKE.

8. For phage annotation, it’s essential that you log into PhAnToMe/BioBIKE. If you’re using a wireless connection and find you’re blocked, try the alternate link.

9. Organization of Project Suppose you want to annotate your favorite genes. Go to the ANNOTATION menu and bring down a VIEW-GENE box

10. Organization of Project Enter the name of the gene by clicking the gene-or-protein box…

11. Organization of Project … which gene? I’ll choose one you’re familiar with. (PhiLC3? Remember that one?) Execute the function.

12. Organization of Project This brings you to the gene’s annotation page. If you want to know something about the gene’s physiological role or regulation, this is the place… …Hmmm. 0? There doesn’t seem to be anything here. This is where you come in. But for now, keep on going. Scroll down.

13. Organization of Project This is a genetic map of the region surrounding the gene, both in PhiLC3 and some other phage. Potentially very useful! Mouse over the red gene marked 1.

14. It tells you that the gene is currently annotated as the phage repressor. (I hope there’s good evidence for that assertion!) Move the cursor a couple of genes to the left Feature ID fig|12390.1.peg.3 Function Phage repressor Contig NC_005822 Start 2781 Stop 1921 Size 861 nt, 287 aa Set 1

15. It says it’s a transcriptional regulator… No doubt! But Blatny et al had a lot more to say than that! You could make significant improvements in this annotation, but for now scroll back up. Feature ID fig|12390.1.peg.5 Function Transcriptional regulator Contig NC_005822 Start 3222 Stop 3452 Size 231 nt, 77 aa Set 16

16. Organization of Project You probably had a difficult time finding the gene during the exam, because the name Orf286 used by Blatny et al was unknown to BioBIKE. You could make things easier on the next person by adding that name to the aliases. To change the aliases, just click on the yellow box.

17. Organization of Project Clicking the box enables you to change its contents. Type in Orf286

18. Organization of Project But as you may have noticed (and if not, you will!), there are all sorts of crazy things in annotations. Every change you made should have a reason. Click the Justification box to supply a reason.

19. Organization of Project That’s fair. The name was used in a published article. But you can’t expect someone else to know what “Blatny et al (2003)” means. You need to provide the full reference. Don’t remember it? Click the PubMed button.

20. Now, with a few choice terms, you can find the article. To bring the reference into BioBIKE, you should be able to just copy the PMID (PubMed ID)… …but that trick isn’t working yet. So for now, get the URL of the article, either by right clicking the article and choosing Copy Link Location, or clicking the article…

21. …and just copying the URL directly from the browser

22. Organization of Project Paste the URL into the Justification Link box…

23. … and click SAVE

24. The change you made has been incorporated into the PhAnToMe data base. If you mouse over the Justification icon…

25. Organization of Project …you’ll see that your change worked. It is now visible to the world. Move the cursor a bit to the right, over the History icon…

26. …and you’ll see the author of the current version of the alias field, also visible to the world. If someone disagrees with the change (or wants to find out more about the reasons), clicking on the name will generate an e-mail to the author.

27. This is how you’ll enter information about the genes you annotate. Your primary responsibility will be towards the genes of either Wally or Batiatus, but you (and anyone else in the world) are free to annotate any gene you know about.

28. Organization of Project Groups 1 through 4 are primarily responsible for Wally, and Groups 5 and 6 are primarily responsible for Batiatus. More on this in a moment, but for now, let’s consider the second part: Analysis. Scroll down to put all the analysis groups on the screen.

29. Phage Phage genome Lysogeny Bacterial chromosome Prophage Organization of Project The Lysogeny Group will consider the protein and DNA binding sites needed by phage to achieve and maintain the lysogenic state. Remember lysogeny?

30. Phage Phage genome Lysogeny Bacterial chromosome Prophage Organization of Project Note that this group (like all of them) comes with a couple of focus questions and a reference (usually a review article) that might help you get started.

31. Phage Phage genome Lysogeny Bacterial chromosome Prophage Organization of Project The Lysis Group will consider the protein and DNA binding sites needed by phage to break the host cell. Killing your host is not a decision to be made lightly. Do it too soon and you kill yourself. Too late, and you lose the race.

32. Organization of Project The optimal time is when the the maximal number of phage particles have been manufactured. John Wertz, E. coli Genetic Stock Center, Yale U.

33. Organization of Project Then the phage synthesizes enzymes that break the host’s cell wall to cause local bursts. What are these enzymes in your phage? It’s up to you to find out and tell the world about it. Ry Young (1992) Microbiol Rev 56:430-481 John Wertz, E. coli Genetic Stock Center, Yale U.

34. Organization of Project The Sequence Bias Group will try to find measures of nonrandomness in the phage genomes. You’ll remember one example from a few days ago: different organisms have different preferences for the codons they use in protein synthesis…

35. Organization of Project Do the phages have the same codon preferences as their hosts? Some phages carry their own tRNAs. Does this influence codon preferences?

36. Organization of Project The most common gene on earth encodes a protein that copies the gene that encodes it and propagates copies of it. Wouldn’t you know it?

37. Organization of Project The Mobile Element Group will attempt to identify such genes and the adjoining DNA sequences

38. Organization of Project A bacterium typically needs to replicate its genome (say 3 million nucleotides) in a leisurely several hours, before the next cell division. Phages are often under much greater time pressure. For example, in the case of phage T4: 300 copies x 170,000 nt per copy = 51 million nt in 30 minutes! With higher DNA synthesis requirements than the host, many phage encode their own DNA synthesis proteins. Mueser TC et al (2010) Virol J 7:359

39. Organization of Project But it’s not all protein. ALL phage DNA need a beginning point, an origin of replication, that determines where DNA synthesis will begin. The DNA Replication Group will look for both phage-encoded DNA synthesis proteins and origins of DNA replication on phage genomes. Mueser TC et al (2010) Virol J 7:359

40. Organization of Project The Gene Regulation Group will look for proteins and protein-binding sites on phage genomes responsible for the regulation of the lytic process. We’ve seen already seen mechanisms that regulate expression of the phage repressor. There are many many other strategies.

41. Organization of Project G D G D All the groups will consider both genes (G) and DNA (D) features. D G G D G D G D

42. Organization of Project All groups will consider how their genes cluster and how the clustering relates to the overall clustering of phage genomes…

43. Organization of Project …and how the clustering might be viewed as an evolutionary tree. All of this takes both analysis of your phage sequences and an appreciation of the analyses others have done before you. How to get that appreciation? You and others in your group will need to look at lots of articles. But lots starts with the first one. In the next two weeks, in consultation with your group, you should identify a useful article, one that broadens the knowledge of your group on your topic.

44. Organization of Project To help you make sense out of articles, you’ll have the opportunity to write a summary of that article… What’s a summary? Click on the link…

45. Organization of Project • Key elements: • Choose a research article (not a review). • Focus on just one experiment. • Understand that experiment and its result to the bone. • Add your knowledge to PhAnToMe.

46. Organization of Project I hope you now see that there will need to be a lot of interaction with your group and focus on your topic. But what is your group? What is your topic? To answer the first, click Analysis Groups.

47. Organization of Project The groups were chosen according to the schedules you provided, to make sure that there was at least one time you all could meet. You can change that time (or supplement it), so long as there is one time that does not conflict with the times of others (so that I can attend the meeting).

48. Organization of Project Each group has been assigned part of a phage to annotate. For example, Group 1 has all the genes that overlap with the first 40,000 nucleotides of Wally. That group will find all genes in that region, determine the functions of the proteins, and put the information into PhAnTome. (We’ll talk more about this) Groups can distribute nucleotides to its members however they like.

49. Organization of Project But which group gets which Analysis Groups?You’ll get details in a forthcoming email, but in brief…

50. Organization of Project • Each group will submit one set of choices. The group must first arrive at a consensus. • Each group will have 10 votes, which it can distribute any way it likes. • An algorithm will take the six group votes and use them to maximize happiness • For example…