MBG 8680: Computer Applications in Molecular Genetics

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INTRODUCTION TO GCG

GCG is a suite of related programs for analyzing nucleic
acid and amino acid sequence data.  The GCG programs are 
installed on the Genetics computer.  This tutorial describes how
to set things up for running the GCG programs and then
demonstrates the use of several useful GCG programs for basic
analysis of sequence data.

BASIC TOOLS NEEDED

To complete this tuturial, you will need to have several basic
tools installed on your PC (or Mac).  These include telnet, 
FTP client, Netscape, text editor, X Windows server, and file 
printer.  If you do not have X Windows, you may run the GCG 
programs through HYBROW (type hy) or from the command line.

DOCUMENTATION

All GCG documentation is available online.  Use Netscape to 
open the GCG manual at:
http://www.genetics.wayne.edu/gcg/gcgmanual.html
The Program Manual is organized by Program Function. 
GenHelp is organized alphabetacally (look up the map program
both ways).

The User's Guide contains general information about GCG.
Data Files contains info about such things as scoring matrices.
Databases contains info about the databases such as GenBank.

LOGIN

Use telnet to connect to genetics.wayne.edu and login.
Determine your IP Address: who | grep userid
Set DISPLAY to your PC: setenv DISPLAY ip.address:0
Start the X Windows program on your PC.
Open the X File Manager: filemgr &     

SETUP FILES FOR GCG

NB: You should complete this section before coming to class.

You will need to copy some demonstration files.  You can
accomplish this from the UNIX command line, as follows:

genetics% cd
genetics% mkdir mbg8680
genetics% cp -r /home/dwomble/mbg8680.template/* mbg8680

Those 3 commands change to your home directory (if you weren't already
there), make the new mbg8680 folder, and finally, copy (-r means
recursively through the folder) all the files and subfolders in my
mbg8680.template folder and deposit the copies into your mbg8680 folder.

After completing the set up, cd into your mbg8680 folder and explore
it's contents to make sure everything worked as expected.

Alternatively, you can accomplish the same result using File Manager:

Determine your IP Address: who | grep userid
Set DISPLAY to your PC: setenv DISPLAY ip.address:0
Start the X Windows program on your PC.
Open the X File Manager: filemgr &

Create a new folder in your home directory called mbg8680
Change into the /home/dwomble/mbg8680.template folder.
Copy the all files and folders.
Change back to your own mbg8680 folder.
Paste in the copies of my files and folders.

START THE GCG PROGRAMS

From telnet, cd into your mbg8680/gcg folder.
List the files in the folder: ls
Start the gcg programs by typing gcg

COPY SEQUENCE FILES FROM THE DATABASE

Use the GCG fetch program to bring back a copy of the m13mp18 sequence
from the database: type fetch m13mp18
(m13mp18 is a single-stranded DNA virus used for DNA cloning/sequencing)
List the files, then look at the m13mp18.gb_sy file: more m13mp18.gb_sy

Note the header information:
Name, Length, Date, Type, Check ..
Note the 2 dots at the end of the header info.  Those are essential 
for dividing the header and the data in GCG format files.
Close the file.

GCG FILE FORMAT AND CONVERSION

Use tofasta to convert a GCG sequence format file into a fasta format
file.  Use fromfasta to convert a fasta format file to GCG format.  
Other formats include staden, embl, genbank, pir, and ig.

PRINTING OR DISPLAYING GCG RESULTS FILES

Use Netscape to open: http://www.genetics.wayne.edu/dwomble/gcggraphics.html.
Read the GCG Graphics and Printing file to learn how to print GCG
results files.

From telnet, type setplot 
Select colorX (note other choices).
Type plottest, examine the test plot, then Exit from the Graphics
window.

SEQLAB, THE X INTERFACE TO GCG

SeqLab is a graphical interface for running GCG programs that 
works through X Windows.  

Click here to see the SeqLab Window.

Start the X GCG Interface: type seqlab &
(seqlab is a GCG program, so gcg needs to be running first;
also, since seqlab is an X Windows program, you need to have
your X Windows server program running on your PC, and have your 
DISPLAY set to the IP address of your PC - see above)
Click Functions; they are organized by program function, like the manual.
Click Alphabetical; the functions are listed alphabetically.

CHANGE THE APPEARANCE OF SEQLAB

You may customize the appearance of SeqLab by editing the SeqLab
configuration file:

In the File Manager, change to your mbg8680/gcg folder.
Copy the SeqLab file.
Change to your home directory.
Paste in the copy of the SeqLab file.
Edit the SeqLab configuration file, then save and close it.
Click File then click Exit to close SeqLab.
From telnet, type seqlab &
The SeqLab file in your home directory can be used to 
customize the way SeqLab looks.

SET THE WORKING DIRECTORY FOR SEQLAB

Files created during your SeqLab session are stored in 
your "Working Directory."

Set your working directory to your mbg8680/gcg folder:
Click Options, Preferences
Click Working Dir
Select mbg8680/gcg then click OK.
Click Apply then click OK.

CREATING A WORKING LIST FILE FOR SEQLAB

SeqLab works by using a list of sequence files.  You 
may create a different list file for each project.  
Here, we create one for this tutorial:

Click File, New List, type mbg8680.list and click OK.

We will now add sequence references to the Working List.  You can add
references from the Databases or from sequence files in your folder.

Click File, Add Sequence From, Databases.
In Database Specification, type m13mp18
Click Show Matching Entries.
Under Entries, click m13mp18, then click View Sequence.
Click Cancel.
Click Add to Main Window, click close.

Note sequence reference added to list (a pointer to the database).
Click File, Extract Sequence.
Click OK (note format choices).
In File Manager, note new file m13mp18.seq (in your mbg868/gcg folder)

In SeqLab, click File, Add Sequence From, Sequence Files
Click m13mp18.seq, click Add.
Click mbg8680.seq, click Add.
Click Close; note new entries in the list.
Click File, Save List.

RUNNING PROGRAMS SUCH AS EDITING AND RESTRICTION MAPPING

NB: You may run programs from either the Main List or from the Editor.

This loads the mbg8680.seq file into the editor:

Click mbg8680.seq to select it.
Click Mode: Main List, click Editor
Note color coded sequence.

Click the sequence to select it.
Click the "i" icon to see information about the sequence, click OK.
Click the padlock icon, check all boxes, click OK.

Move to the 3' end of the sequence.
Click the cursor on the 3' end.
Type in a few ACGT, then delete them.
Move back to the 5' end.

This creates restriction and translation maps of mbg8680.seq:

Click Functions, Mapping, Map (note options), click Run.
Click Windows, Job Manager (note jobs).
Click Open Output Mgr (note result file xxx.map).
Display the .map result file, then click Close.
Close the Output and Job Manager Windows.

Click Functions, Mapping, Mapsort
Click Enzymes, scroll to BglII, click it, click Add
Click Close, click Run.
Click Windows, Job Manager, click Open Output Mgr
Click Display, click Close, close Output and Job Manager Windows.

PRIMER SELECTION

This predicts primer oligonucleotides for mbg8680.seq:

Click Edit, Select Range; type 300 for the End.
Click Select, click close.
Click Functions, Primer Selection, Prime
Click Selected region.
Note Options, but just click Run.
Click Windows, Job Manager, click Open Output Mgr

Note 3 Output files: .log     info about your job
                     .figure  graphical display file
                     .prime   details about your primers
Close the Output and Job Manager Windows.

TRANSLATION

This translates an ORF from mbg8680.seq into a peptide sequence:

Click Edit, Select Range; click Clear, set Begin at 214, end at 1176.
Click Select, click Close.
Click Functions, Translation, Translate, Selected region.
Unclick List file of output sequence names, click Run.
Click Windows, Job Manager, click Open Output Mgr
Display the .translate sequence file, then close the Display.

Click Add to main list.
Close the Output and Job Manager Windows.
Click Editor, Main List, Don't save.
Note new .translate sequence file was added to main list.
Click file, Save List.


REFORMATTING FILES INTO GCG FORMAT

In this section we will use the Hybrow menus to control the 
GCG programs.

If not already open, open a telnet session to Genetics and start the 
GCG programs by typing gcg.  Start the Hybrow menus by typing hy.

This creates a plain text sequence file:

From the Hybrow main menu, go into dirctory browser.
Move into your mbg8680/gcg folder.
Go back to the main Hybrow menu.
Select pico editor to edit a new file.
Type some bases, ACGTACGT etc.
Press Ctrl-X (^X) to exit the editor.
Answer Yes to save the changes.
Type new.txt for the filename to save, press return.

This reformats the text file into GCG format:

From the main Hybrow menu, go into HYGCGmenu, then
press return to get to the main GCG menu.
Go into Convert Sequence Format, then into Reformat.
Point to the file new.txt, then press x to execute reformat.
Press return to accept all default paramaters.
Press v to view the new.txt file, note GCG format.
Press q to quit viewing the file.
Press r to rename the file, type new.seq for the name.

You can convert any plain text sequence file to GCG format by this 
method.  You can also do the same from the command line or from 
within SeqLab.

To convert from one sequence format to another, use the to and from
utilities.  For example, to convert a FastA format file to GCG
format, use fromfasta.  To convert a GCG format file to FastA 
format, use tofasta.
Send comments to: dwomble@genetics.wayne.edu
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