Assignment 2

The Multiple Loci VNTR (variable nucleotide tandem repeat) analysis (MLVA) technique is used to identify strains of organisms.

Because the database is quite large for S. aureus and publicly available, we're going to focus this second assignment on an analysis of this method. Go to the website http://mlva.u-psud.fr/mlvav4/genotyping/index.php, which contains a link to a number of databases. On the left menu, click on the Staphylococcus_aureus database under the Collaborative databases option. First click the "View" button. You'll then see appear a large list of different isolates of this strain.   Click on the "Panels" button on the right to reveal different MLVA data. 

1. Under the MLVA section to the right, there are a series of mostly tags: "Sa0122, Sa0266", ….   Below that are mostly integers. Explain what these tags mean and what the numbers stand for, based on your readings in other sections this week.

2. Describe the difference between MLVA and MLST.

3. Why are some of the MLVA numbers blank?

Now go back to the left menu and select "Staphyloccus_aureus" and choose the "Query" button instead.

4. There is a button that says "select a panel", or you can select using checkboxes the ones you want to use. Describe the difference between the two.

Select the MLVA14Orsay option and click "Submit".  (make sure it is the MLVA option on the left, not the MLSA on the right).

Now you see a MLVA box listing the different loci that you are going to query. Each site is a different MLVA marker. Below is an option where you can enter your MLVA set with a space between each number.   Notice that there are 14 numbers, matching the MLVA14Orsay option. Type in the sample sequence exactly as listed to see what the sample output would look like. "10 6 3 4 3 7 7 5 4 4 5 2 3 3"

You get a direct hit (of course), and it shows you the those fingerprints most closely related to the one you chose. To which strains was this hit most similar?

5. Does this mean the strains themselves came from these locations?      

Notice how the red boxes indicate differences from the sequences that you entered.

6. Click on "Tree" using the "Tools" pullout menu, and select the Newick tree.  Download the tree as a text file.  Upload it here (http://etetoolkit.org/treeview/) and download the graph that you get from it, placing it into this assignment. 

Now we're going to switch over to doing analysis of Yersinia pestis using MLVA typing. Go back to the main page and select "Yersinia pestis2004". Again, click "View" so you have an idea of what you are looking at.

7. The nomenclature is slightly different, as are the markers. Why?

8. Why doesn't it make sense to use 50 markers for MLVA analysis, to increase specificity? Here's a paper that discusses the addition of more VNTRs the current Anthrax MLVA analysis for ideas. http://www.ncbi.nlm.nih.gov/pubmed/19469279

9. Open the Y. pestis MLVA7 database and choose one of them.  (For example, I chose (10 9 9 8 7.5 9 6).)  Make a tree using the instructions on the website.  Purposely make a mismatch and see what happens when you generate a second tree.  What changes?  Describe the major difference between the two "trees".

10. Go back to the main menu. Select "Geographic View". Notice how it takes you to a world map that shows you where the isolates were found.

11. Access the matrix of distances. What does this do?

12. Find at least one other MLVA site that does sequence analysis and post it in the conference area. In one or more sentences, briefly say what the site is, what it focuses on, and what nation it is from.

13. Complete your assignment by summarizing what you've learned in one paragraph.  It should be a rather detailed paragraph that reflects on the various issues that this assignment has shown you.

Here are a few more sites that talk about some issues with this database.

http://mlva.u-psud.fr/MLVAnet/spip.php?article90

APA format, in-text citation, references include, 3 pages to answer all 13 questions.