AlignX for protein alignments

The AlignX program, part of the vectorNTI suite can be used to perform protein and DNA sequence alignments. In this section we will align some sequences from the database

Start vectorNTI, and in the Database Explorer select protein molecules. Then choose the four sequences named GNAI1, GNAQ, GNB2, and GNB5

A new window opens with the AlignX program.

AlignX

AlignX allows you to perform multiple pairwise alignments. To begin, select a few sequences. For example, scroll down the list somewhat until you find the series of TNFA sequences. Select just those sequences by clicking on the first one, and shift-clicking on the last one in the list

You can change the parameters for the alignment by selecting Alignment Setup from the Alignment window. Parameters used for alignments, and what they mean will be discussed futher in module 7 and will not be discussed here.

Once you have selected a few sequences, choose Align Selected Sequences from the Alignment Menu

The resulting view is split into 6 panes.

1. The text pane contains all the sequence information.
Clicking on the plus sign will expand the box showing all of the information about that sequence that was acquired from the vectorNTI entry. You can highlight a particular entry (such as a Feature) by selecting that entry and then clicking on the Find icon: The region that you have selected will be highlighted in the graphical view and the alignment view as shown below.

2. The Graphical view contains 3 panes. Each demonstrates some facet of the alignment:
1. The Similarity   is a score of how similar each amino acid (or groups of amino acids) are across the whole alignment.
2. The Absolute Complexity   is the average of the pairwise alignment scores using the substitution matrix chosen in the alignment setup.
3. The Absolute Complexity   of an individual sequence is the pairwise alignment score between the selected sequence and the consensus sequence. The sequence may be selected by clicking on the sequence name in either the tree view, or the alignment pane. Additional analyses can be added by clicking on the
Each of the views in this window may be edited by right clicking on the view and selecting Plot Setup. This brings up the set up displays:



• The Datagram allows you to select polygons, lines, etc, and change the color of the datagram.
• The Analysis allows you to set the size of the analysis window. By default this is 5 residues, however you can increase or descrease this number. The point that is plotted uses this as a sliding window that is incremented along the sequence.

A region of the sequence may be selected in any of the plots, and this region will be higlighted in the sequence shown in the Sequence Alignment Pane.

3. The Phylogenetic Tree Pane  
The phylogenetic tree can be used to infer an evolutionary relationship between the sequences in the alignment. This tree is built using the Neighbor Joining Method (which will be described in module 8). The tree is only present if three or more sequences are used in the alignment.
4. The Sequence Alignment Pane  
This pane shows the alignment of the sequences, color coded according to similarities. The default settings are:
• Identical   Red text on yellow background
• Weakly similar   Dark green text on a white background
• Block of Similar   Black on a light green background
• Conservative   Dark blue on a light blue background
• Non-similar   Black on a white background

The settings and sequences in the alignments may be changed:

• To edit the sequences click on the Edit Alignment button:
This button will bring up the editor dialog:



Sequences can be moved by highlighting a stretch of sequence and then clicking either the move left or right buttons. Note that sequence can only be moved when there is an adjacent blank space (denoted by a "-").
• To remove a sequence from the alignment, click the Remove From Alignment button:
Note that this will not delete the sequence from the current set of sequences shown in the text pane, but just remove it from the alignment. To delete a sequence entirely, click on its name in the text pane, and then right click and select Del From Project.
• To edit the set up either right click and select Display setup or click the Display Setup button:
This opens three menus shown below:



• The Consensus Calculation allows you to set the parameters for the consensus that is shown below the sequence and used in the analysis of complexity in the graphics pane.
• The Similarity Table determine which residues are colored as similar or not in the alignment
• The Color Setup allows you to determine which colors are used in the alignment shown for each of the groups mentioned above

Summaries

Two simple summaries of the alignments are available from the Alignment menu.

1. Percent Difference and/or similarity

This will display a table with the percent similarity and/or difference between the pairs of sequences in the alignment. In the example shown here the top right matrix contains the percent similarities and the bottom/left matrix contains the percent differences



2. DotPlots are pairwise comparisons with one sequence on each axis. Conserved sequences are marked by a dot, and hence the lines that form in the plot represent runs of contiguous sequence that are similar to each other.

   

   Other types of alignments

   Several other types of alignments are available with the alignX package:

   • Align Selected Using Profile will use one of the sequences in your group as a profile on which to base the alignment. This will optimize the alignment towards that particular sequence, and is useful for comparing a known sequence to unknown sequence(s).
   • AlignX Blocks aligns sequences in blocks. This aligns short regions of sequences without gaps and generates them into blocks of sequence. This is useful for working with proteins that form domains. AlignX blocks is not described further right now, but may be if there is requests.