Phred/Phrap and Consed Workshop Protocol

The original document (MSWord) can also be downloaded

1. Download Putty

1. Turn on your computer

-Login: classroom Password: ********

2. Go to the “My Computer” icon and double-click on it. In My Computer go to Mlab onCl1-mlab… It should be the first folder in the Network Drives section.

3. Next go to the SHARED folder and scroll to the bottom of the folder.

4. Double click on putty.exe.

5. In the “Host Name” box type “capecod.bwh.Harvard.edu”

6. Right below the Host Name box select the SSH function.

7. In the “Category” field to the left click on Tunnels under SSH.

8. Check the Enable X11 forwarding box and press “Open” located in the bottom right.

9. Login as: rewrk Password : ********

2. Open X-Win32

1. Go to the Start Menu and go to the telnet and other applications menu. select the XWin32 program. XWin32 should open automatically and no further instructions are needed.

3. Copy Chromatogram File Folders

1. In the putty console type on the Capecod sun server: “cd ~reseq00/PGAworkshop”

2. Next type “ls to find what is available on the UNIX system within this directory.

3. Type “mkdir ‘your last name’”

-This creates a directory that is named your last name. You will work within this directory for the rest of this workship

4. Copy the AQP5 and the tarc folders into your personal folder by typing:

“cp –r AQP5 ‘your name’”

- do the same for the tarc folder.

5. Type cd ‘last name’ to go into the folder you just created.

6. Type “ls”

-You should see both the AQP5 and tarc folders.

4. Create 4 Phred/Phrap Folders

The Phred/Phrap programs require these folders to be present.

1. Type “cd AQP5” and then type “ls”

-A chromat_dir folder should appear.

2. Make your directories by typing:

“mkdir edit_dir”

-Make directories phd_dir and poly_dir in the same manner

3. List your folders by typing “ls”

-Listed folders should be: chromat_dir, edit_dir, phd_dir and poly_dir

5. Create a Reference sequence

1. Go into your chromat_dir folder by typing “cd chromat_dir”

2. List samples in the file by typing “ls”

-All of your chromatograms from the ABI sequencer should be stored here as well as a file named AQP5refseq.txt.

-The .txt file is your fasta reference sequence obtained from the golden path database at UCSC.

3. Create a Phred/Phrap friendly reference sequence by typing:

“fasta2Phd.perl AQP5refseq.txt”

4. List your files in your folder (type ls)

-You should now see a file named AQP5refseq.phd.1

5. Move the created phd file to your phd_dir folder by typing:

“mv AQP5refseq.phd.1 ../phd_dir”

6. Running Phred/Phrap

1. Now that you have constructed a reference sequence it is time to run phred/phrap. Change directories to your edit_dr folder by typing “cd ../edit_dir”

3. To run phed/Phrap type “phredPhrap.longreads”

4. When prompted, type consed.

7. Using Consed to View Assemblies

1. Double click on the ace file created.

2. A consed window should appear. Open the only file available in the Contig List by double clicking on the file.

3. Another window should appear. Maximize the new window by pressing the square button located at the top right.

4. Uncomplement your sequences by pressing the Compl Cont button located at the bottom of the toolbar.

5. View individual sequence tracings by middle clicking on a base. Note: you can not view your reference sequence as it does not have a chromatogram.

8. Runing Polyphred For Variant Detection

1. Press the Dismiss button located on the bottom right hand corner of your contig.

2. Close Consed by pressing the Quit Consed button.

3. When prompted to save your work and in the new window that opens press OK

4. In the Putty console type “ls”

-The files listed were generated by Phred and Phrap

5. Run Polyphred by typing “polyphred –ace AQP5.fasta.screen.ace.2 > AQP5.polyphred.out”

6. When the program is finished running open consed.

7. Open the contig file. Note the Yellow labeled sequence and the purple labeled sequence.

8. Confirm and Tag a polymorphism

   1. At approximately the 180 base pair position of your consensus sequence, there should be a possible SNP labeled in purple. View the tracings to confirm the SNP.

   2. Tag the SNP by middle clicking on the consensus sequence.

   3. A window should appear named “Select Tag Type”

   4. Double click on Polymorphism.

9. View and clean tagged polymorphisms.

   1. select Navigate in the toolbar and selecting Tags.

   2. In the Tag window that opens select Polymorphism from the list by double clicking on it. A list of tags should open.

   3. Go to the beginning of exon 5 at approximately position 4450 of your consensus sequence (sequence tracings named AQP5X_E5F_00NN).

   4. Note the 2 possible polymorphism and the “bad” sequence tracings that flank the polyphred tags.

   5. Compare the sequence to a good sequence trace by middle clicking.

-The bad sequence is actually an insertion.

1. Tag the insertion by middle clicking on the consensus sequence and select indelSite from the menu.

2. The insertion is now stored in Tags section of navigate under indelSite.

1. Quit Consed

9. Viewing Your Export Data

1. In the putty console type “more AQP5.polyphred.out”

2. You should see two sections in the text output. The first are the polymorphisms polyphred has tagged, the second section contains the tags you have manually selected.

3. Press enter to see more of the data or press “q” to exit the export file.

10. Analyzing the TARC data

1. Type “cd ../..” to get you back to your main folder.

2. Type “cd tarc” to enter the tarc folder.

3. Follow sections 4, 5 and 6 replacing AQP5 with tarc.

4. Type “consed” when prompted to do so

5. Double on the ace file created.

11. Editing Assemblies

1. Two contigs should appear, open Contig 1. The sequence should have a green strike through most of it. This indicates a match in another contig of the same sequence.

2. Select approximately 20 base pairs of sequence by pressing the left hand button on the mouse and dragging. You should see the sequence become highlighted.

3. In the left hand portion of the toolbar press the Search for String button.

4. Middle click in the empty box, your sequence should paste and press OK. If there is not a middle mouse button, then click both buttons at once.

5. A window should appear with 2 contigs listed. Double click on contig 2 to open contig 2.

6. In the tool bar of each alignment window press the Compare Cont button.

7. In the Compare Contigs window press Align. The two contigs line up pretty well.

8. Press the Join Contigs button to finalize the process and press OK when the warning window appears.

9. In the main contig window notice that contig 1 and contig 2 are replaced by contig 3.

10. Quit consed and save.

11. Editing by Increasing PhredPhraps Forcelevel

This forces Phrap to assemble sequences more liberally. There are other forcelevels and should be used iteratively as it creates more manual editing of polymorphisms.

1. In the putty console type “phredPhrap.longreads –forcelevel 5” and when prompted, type “consed”

2. Notice that there is only one contig.