introduction to bioinformatics wednesday, 2 march 2011 genome analysis hatfull et al (2008) break up...

Introduction to BioinformaticsWednesday, 2 March 2011

Genome analysis

• Hatfull et al (2008)• Break up into research groups

• Organization of research groups

• Introduction to Wally and Batiatus

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Viral Genomes Project

Viral Genomes Project

Wally Batiatus

Rachel Walstead and Danielle Renner


Genome analysis




Organization of Project

The organization of the project can be found on the Genome Analysis module page


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)…

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.

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.


Suppose you want to annotate your favorite genes. Go to the ANNOTATION menu and bring

down a VIEW-GENE box


Enter the name of the gene by clicking the gene-or-protein box…


… which gene? I’ll choose one you’re familiar with.

(PhiLC3? Remember that one?)

Execute the function.


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.

Organization of ProjectThis 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.

Feature

ID fig|12390.1.peg.3Function Phage repressorContig NC_005822Start 2781Stop 1921Size 861 nt, 287 aaSet 1

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.5Function Transcriptional regulatorContig NC_005822Start 3222Stop 3452Size 231 nt, 77 aaSet 16

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.


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.


Clicking the box enables you to change its contents. Type in Orf286


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.


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.

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…

…and just copying the URL directly from the browser


Paste the URL into the Justification Link box…

… and click SAVE

The change you made has been incorporated into the PhAnToMe data base.

If you mouse over the Justification icon…


…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…

…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.

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.


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.


Lysogeny

Phage

Bacterial chromosomePhage genome

Prophage

The Lysogeny Group will consider the protein and DNA binding sites needed by phage to achieve and maintain the lysogenic state. Remember lysogeny?


Lysogeny

Phage


Prophage

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.


Lysogeny

Phage


Prophage

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.

Organization of ProjectThe optimal time is when the the maximal number

of phage particles have been manufactured.

John Wertz, E. coli Genetic Stock Center, Yale U.

Organization of ProjectThen 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.

John Wertz, E. coli Genetic Stock Center, Yale U.

Ry Young (1992) Microbiol Rev 56:430-481

Organization of ProjectThe 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…

Organization of ProjectDo the phages have the same codon preferences as their hosts?

Some phages carry their own tRNAs. Does this influence codon preferences?

Organization of ProjectThe 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?

Organization of ProjectThe Mobile Element Group will attempt to identify

such genes and the adjoining DNA sequences

Organization of ProjectA 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

Organization of ProjectBut 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

Organization of ProjectThe 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.


All the groups will consider both genes (G) and DNA (D)

features.

GD

GD

GD

GD

GD

GD

Organization of ProjectAll groups will consider how their genes cluster and how

the clustering relates to the overall clustering of phage genomes…

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.


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…


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.


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.


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).


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.


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


• 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…


Your group might cast 7 votes for Gene Regulation, and 3 for Mobile Elements. If your group has the highest number of votes for Gene Regulation, you’ll probably get it.

But if you cast all of your 10 votes for Gene Regulation, and another group has done the same, then that group might get Gene Regulation, and you will be probably be given whatever’s left over, probably an analysis group that attracted little interest from other groups.

(Not sure how best to achieve your ends? You might try to simulate the game.)


Genome analysis




Hatfull et al (2008): Protein families

Hatfull Graham F, Cresawn Steven G, Hendrix Roger W (2008) Res Microbiol 159:332-339


Genome analysis




Analysis Groups (Geography)

Group 1

Group 2

Group 3

Group 5

Group 6

FRONT

Group 4

introduction to bioinformatics wednesday, 2 march 2011 genome analysis hatfull et al (2008) break up...

Documents

batiatus slide

left slide

organization of project

viral genomes project

viewgene box slide

protein box slide

danielle renner slide

phage annotation