EXERCISE 1.

In this exercise you will use the program Sequencher to assemble a series of overlapping sequences of the gene PKDREJ from the chimpanzee. You will compare the chimpanzee sequence to the human sequence and check for differences. The main goal of the exercise is to get a feeling of how gene sequences are obtained. Links to sequence files and programs are at: http://faculty.washington.edu/wjs18/MolEvol.html

Sequencher.

1. Download sequence files from website.
2. Use the UCSC Genome Browser or NCBI to find and download the human PKDREJ gene.
3. Import abi sequence files and human reference into Sequencher.
4. Assign the human sequence as the reference sequence.
5. Check and edit each individual abi sequence file. Remove regions with lots of Ns and low quality sequence. This can be done using "trim ends" command under the sequence menu, or do each one manually. Trim the ends of a few manually first, then try "trim ends" command.
6. Sequence files pt13.abi and pt14.abi were cloned into a vector. You need to trim off the vector sequence. The vector sequence is:

GAA TTC GCC CTT - AGG GCG AAT TCT

CTT AAG CGG GAA -TCC CGC TTA AGA

The - indicates insertion site.

7. Select all sequences, then assemble the contig by "assemble automatically".
8. Compare regions that are different. Find some changes that look reliable.
9. Edit sequences until contig is complete. You may not finish, just get as far as you can. You can not save work because we are using a demo version.

Questions.

1. Get a screen shot of a region where there is a real difference between human and chimpanzee sequence. Describe why you feel this is a real difference.
2. Get a screen shot of a region where two of the chimpanzee sequences disagree. How did you decide which is the correct base call?
3. Get a screen shot of the "overview" of the contig showing the coding region: did you get a complete open reading frame?
4. Give a brief description (1-2 paragraphs: ~1/2 to 1 page) of what you did during the lab section to assemble the chimpanzee PKDREJ sequence. What problems did you have?