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SeqLab Tutorial

To start working with SeqLab, first startup your X-windows program, then login to your account on Ranger by telnet and start GCG. Then type seqlab

After a few seconds you should see a "Welcome to GCG" splash screen and the SeqLab Main List.

All of the GCG programs are available from the Functions menu.

This tutorial project will be to make a multiple alignment followed by building a phylogenetic tree.

Build a list of sequences

First, use FASTA to generate a quick list of sequences to align.

Lets start with the human rab11 protein. Add it to your Main List by clicking on the File menu and choosing "Add Sequences From > Databases..."

In the new window that appears, click on SwissProt in the list of databases at the upper left, then enter the accession number P62491 in place of the "*" in the text field at the bottom of the window. Then click the "Show Matching Entries" button at the upper right. Once the sequence "swissprot:p62491" appears in the center column, click on it to select it, then click the "Add to Main List" button at the bottom left of the window.

Now lets use the p62491 sequence as a query in a FASTA search of the Swisprot database. From the Functions menu, choose "Database Sequence Searching > FASTA..."

When FASTA is done, the Output Manager window will open automatically as well as a window with a log file and a window with the actual text of the results file created by FASTA.

You should have a long list of proteins that are similar to rab11.

In the Output Manager window, select the new .fasta file and load it into the Main List (click the button "Add to Main List" on the right side of the window).

Close all windows except the Main List.

Double click on the p62491.fasta file in the Main List and it will open up into a very long list of all sequences that were found in the FASTA search.

With the mouse, highlight the first 27 sequences in the list (down to R11C_TOBAC), then at the top of the window, click on the "Mode" button to change it to "Editor". If your "Display" is set to "Residue Coloring" you should have a very colorful screen full of sequences.

Make a multiple Alignment

Now use PILEUP to make a multiple alignment of these proteins.

Use the mouse to select all of the sequence names in the column at the left side of the screen.

From the Functions menu choose Multiple Comparison and then PileUp.

Use all the default settings, so just click the Run button at the bottom of the PILEUP window.

When PILEUP is done, SeqLab will automatically display the figure file in a graphics window and the multiple alignment output in a text window.

In the Output Manager window, select the pileup.msf file created by PILEUP and load it into the Main List (click the button "Add to Main List" on the right side of the window).

Close all windows except the Main List.

Do not worry about loosing the figure - SeqLab keeps all output files in the Output manager until you specifically delete them.

Use the Editor to view the Multiple Alignment

Now in the Main List, select the pileup.msf file, then from the "Mode:" button, choose Editor.

You will see a notice that SeqLab is loading sequences into the Editor, and then your multiple sequence alignment will appear in multicolored glory.

You can adjust the alignment as you see fit by adding and deleting gaps or rearranging sequences in the list (using the cut and paste buttons).

Click on the sequence name in the left-hand column to work with an entire sequence, click on a single sequence character or drag over a range of characters to work within a sequence (or group of sequences).

Build a Phylogenetic Tree

Once the multiple alignment is adjusted, use the Evolution tools to create a phylogenetic tree.

From the Edit menu, choose Select All.

Then from the Function menu choose Evolution and then Distances.

Click on the Kimura distance method and also GrowTree (this will automatically create the tree once the pairwise distances are calculated by DISTANCES).

Then click the Run button.

From the Output Manager you can call up the Pileup.figure to compare to the figure produced by GrowTree.

SeqWeb

Now try to repeat this same exercise using SeqWeb. It is a lot easier, so I won't give step-by-step instructions, but take note of where you have fewer options and less control.

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Using Computers for Molecular Biology
Stuart M. Brown, Ph.D, RCR, NYU Medical Center

Comments to: browns02@mcrcr.med.nyu.edu