PART 3. VISTA excercises.

Download and save the following files to your directory (use your browser's "file / save as"):

• Human Sequence (human_srf.fasta)
• Mouse Sequence (mouse_srf.fasta)
• Human Annotation (human_srf.exons)

Examine the files; the description of the contents of the files can be found by clicking on the "sequence" and the "Annotation" links on the VISTA input form.

Fill out the VISTA input form. Your base organism will be human, the second organism will be mouse, and the plot title should be "Ex3A". Skip the third organism, window length, conservation length, and minimum length -- the defaults will work fine. Enter the correct files in the sequence and annotation boxes, then wait for your results to be emailed to the address you entered. When looking at your emailed results, it may be necessary to *right-click* the link provided, and select "Open link in new window", in order to get a display.

Question: What do you see in the plot?

Question: What do the peaks signify?

Question: Which areas are conserved exons?

Question: Which areas are CNSs?

Question: Which areas are Introns?

Question: Which areas are Gaps?

B) Download and save the following files to your directory:

• Human sequence (human_50k.fasta)
• Mouse sequence (rc_mouse_50k.fasta)
• Annotation (human_50k.exons)

Examine the files. Enter the correct files in the sequence and annotation boxes, and click Submit to get the results, again by email, after a few minutes.

Fill out the VISTA input form as before; title the plot "Ex3B".

Question: What do you see? Does this mean that there is no homology? Let us try something different.

C) Fill out the form just as in B, but this time, enter the name "Ex3C" and select (highlight) "human/mouse" in the "reverse-complement in base/second alignment" box.
Enter the correct files in the sequence and annotation boxes, and click Submit to get the results, again by email, after a few minutes.

Identify the regions of high homology.

Among these, Identify the exons, UTRs and CNSs (conserved non-coding sequences).

Question: Which of these regions correspond to known human genes?

Consider some reasons there are highly conserved regions outside of known genes.

D) We will now look at a three-way alignment.

Download the following files:

• Human Sequence (human.fasta)
• Mouse Sequence (mouse.fasta)
• Dog Sequence (dog.fasta)
• Annotation (IL4.exons)

Fill out the VISTA submission form. Your base organism will be human, the second organism is dog, and the third is mouse. Name the plot "Ex3D". Enter the correct files in the sequence and annotation boxes, and click Submit to get the results, again by email, after a few minutes.

When specifying the closest organisms for the RepeatMasker, select "cow" for the second organism.

The results will be returned in PDF format. You will notice that instead of one alignment, you now get 3 -- human/dog, human/mouse, and dog/mouse

Question: Can you see peaks that are present in all three alignments?

Question: Can you see height differences among these peaks?

Question: What does height difference mean?

Question: Can you see peaks that are present in just one alignment?

Question: Which pair of organisms is most similar?

Question: What additional information can you gather from a three-way alignment?