1. The dot matrix analysis deserves some special attention. Choose Analysis ➧ New Analysis..., choose to do a protein analysis, select the Dot Matrix analysis, and click the OK button.
2. Click on the Input Sequence icon along the left of the setup panel. As your two sequences, select the proteins Drosophila hsp22 and Drosophila hsp23 (they are both in the Dros Hsps peptide file).
3. Select Dot Matrix on the left of the Analysis Setup Window and examine the panel that appears (Figure 41). Window size is the length of the sequence segments that will be compared - set this to 20. Select the PAM40 table in the Table popup. This table is a scoring table that indicates how similar two amino acids are to each other. In the Threshold box, enter 40 and then press the Add Threshold button. Repeat this procedure to add thresholds of 20, 0, -20 and -40 by typing in the number in the Threshold box and then pressing the Add Threshold button. These values appear in the Thresholds list at the right.

   Figure 41: Dot Matrix Setup Panel

    

4. Set the Dot size to 2 x 2 using the small popup menu in the lower right. This sets the size of the dots to be drawn in the plot to be 2 pixels on a side.
5. Colors now need to be defined for each threshold. Select the 40 in the threshold list and make it red using the Format ➧ Color menu. Next choose the 20 and make it magenta. Make 0 green, -20 cyan, and -40 blue. Note how the Color Ranges "thermometer" on the right reflects your changes. Start the analysis by choosing Run .
6. When the dot matrix analysis is run, the actual comparison data is saved with the output object, so changes in the display can be made easily and quickly after the analysis is completed. This allows you to fine tune the display to show exactly what you want. Your initial output should look like Figure 42. Each dot represents a point of similarity between the two sequences.

   Figure 42: Initial Dot Matrix Output

    

7. Double-click on the dot matrix analysis object to make it the target and then choose Object ➧ Reformat.... Select the Threshold Settings icon on the left to see a panel similar to what you saw in the initial setup (similar to Figure 41). You cannot change window size, but you can change the thresholds and/or their colors. By redefining the thresholds, you can present the data differently. This is useful for reducing the noise level in the plot and highlighting the data of interest.
8. Select the 20 in the threshold list and type a 30 into the threshold box; press the Change Threshold button to change the 20 to a 30. Next select the 0 from the list and change it to 20 by typing in a 20 in the threshold box and pressing Change Threshold. Convert -20 to 10 and -40 to 0 using the same procedure. You have now changed the thresholds from 40, 20, 0, -20, -40 to 40, 30, 20, 10, 0 and left your color scheme intact. Press Reformat and notice how this changes the display. This reformatting ability lets you "play" with the dot matrix results and find the best settings to display your data.

   Figure 43: Selecting a Region in a Dot Matrix Plot

    

9. Double-click the dot matrix output object to make it the target.
10. Use the mouse to drag a rectangle within the plot from about the middle of the plot down to the lower right corner, selecting the diagonal line, as shown in Figure 43. You have now selected a range of each sequence that shows similarity in the dot matrix plot.
11. Choose Object ➧ Align Selection.... This allows you to do a sequence alignment directly from the dot matrix plot. Set up the alignment parameters to match those shown in Figure 44 and then Run the alignment. This will generate an alignment of the regions of the two sequences you selected in the dot matrix plot.

Note that you have just used an output object to define and launch another analysis. With this approach, you can explore your results in an intuitive and flexible way. A number of analyses allow you to use the displayed data as the starting point for other analyses.

This concludes this tutorial. You may quit or continue on to the next tutorial. If you choose to continue, close all open windows now.