It is often desirable to have the results of an analysis directly connected to the sequence being analyzed in such a way that if the sequence is changed, the output object is recalculated automatically without you having to remember to do it manually. The sequence might represent one that you are refining in the lab, a multiple aligned consensus sequence, or a contig. The Gene Inspector provides this ability through hotlinks as described in this tutorial.

1. Open the DNA sequence file called rhodopsins.
2. Choose File ➧ Save As… and save this sequence document as rhodopsins2 in a location on your hard disk that you will remember. You will be making some changes in the sequences in this document and do not want to accidentally change the original sequences.
3. Click on the sequence name lamprey rhodopsin in this sequence window to select that sequence.

   Figure 12: Simple Base Composition Setup

4. Hold down the shift key and then click on the xenopus rhodopsin sequence name. This is a standard method for extending a selection; in this case we are extending our selection from just being lamprey rhodopsin to being lamprey, octopus, and xenopus rhodopsin.
5. Leave these three sequences selected in the sequence document and choose Analysis ➧ New Analysis… to start a new analysis. Choose to perform a nucleic acid analysis and then select Base Composition . If you are not sure how to do this, refer to Tutorial 3: Using Analysis Setups. For our purposes in this tutorial, we will do only a simple mononucleotide composition as a graph, as shown in Figure 12.
6. Because the sequences were selected in the sequence editor document, they are already entered as sequences to be analyzed, as shown in Figure 13. If you had selected a range of nucleotides in a sequence, that range would be indicated and the Segment button would be “on”.

   Figure 13: Sequences Are Already Chosen

7. Run the analyses by pressing the Run button.
8. Arrange and resize the three analysis objects to all fit on the screen for easy viewing and then select them all by selecting one and choosing Edit ➧ Select All.
9. With the three analysis objects selected, choose Notebook ➧ Links ➧ Automatic. You will see the appearance of a small green circular “adorner” icon in the top right corner of each output object indicating that it is now hotlinked (Figure 14). They should all be green, indicating that no sequences have been changed since the analysis was initially run.

   Figure 14: Hotlinked – No Updating Needed

10. Bring the rhodopsins2 sequence window to the front again (you can use the Windows menu to do this). Click in the Xenopus sequence and type in a few characters. This changes the sequence and will notify the corresponding output object in the GI notebook that it needs to be updated.
11. Bring the notebook window to the front and notice the change in the hotlink adorner for the analysis that depends on the Xenopus rhodopsin sequence. The adorner now is red and yellow containing an exclamation point as shown in Figure 15. Notice that only the analysis object which is dependent on the altered sequence needs to be updated. The other two analyses remain OK.

    Figure 15: Hotlinked Object Needs Updating

12. To update the analysis, choose Notebook ➧ Links ➧ Perform Auto Recalc Now… . You should then see the dialog shown in Figure 16. Click the Recalculate Now.

    Figure 16: An Auto Recalculate Dialog

13. Save the notebook and close it. This will save all of the analyses in their current state, including the Xenopus base composition based on the sequence in the currently open rhodopsins2 sequence file.
14. Close the rhodopsins2 sequence document but do not save the changes . This will save the sequences in the state they were in before any changes were made during this session.
15. Open the notebook you just saved. You should see a dialog like the one shown in Figure 16. This happens because the analysis object in question was created using the changed Xenopus sequence, but the rhodopsins2 sequence file was saved without the changes made while the document was open. Thus, the version of the sequence used in the analysis is different from the version of the sequence saved in the file. This means that an update is needed.

Hotlinks can be very useful. For example, you might create a notebook containing many analyses all hotlinked to a specific sequence. When you want to perform this set of analyses on a new sequence, just paste in the new sequence in place of the original sequence and then perform the auto recalc.

This concludes this tutorial. If you choose to continue to the next tutorial, close all open windows now.