This section of the quick tour explains how you can quickly move to the interesting regions in a contig. What is interesting will depend on the kind of project you are doing, so CodonCode Aligner allows you to define "features" (also called "regions of interest"). You can define a variety of different things as features, for example gaps in sample or contigs, discrepancies, low coverage regions, or tags that have been added by programs like PolyPhred. After defining features, you can:
The example we describe here assumes that you have done all the steps (base calling, end clipping, vector screening, and assembly) exactly as described in the preceding sections of this Quick Tour.
From the "Go" menu in CodonCode Aligner, select "Define Features...". This will open the preferences dialog with the "Features" panel selected:
Leave most selections unchanged, but make sure the checkbox "Any discrepancy" is checked. Then, press the "OK" button.
Now open the contig view for the contig in our "Example1" project (from the previous steps in this Quick Tour). Select the "Go" menu. The item we want to use is "Next Feature". Notice the keyboard shortcut shown in the menu - it is Command-Right arrow on OS X, and Control-Right arrow on Windows. Similar, the shortcut for "Previous Feature" is Command-Left arrow respecively Control-Left arrow.
Try the "Next Feature" keyboard shortcut out from the contig view. You will notice that the the cursor and selection in the contig view move to the right, sometimes by a few bases, sometimes by many bases. To see what kind of feature you just navigated to, look at the project view window. The status panel at the bottom shows a brief description of the feature (in our example, "Feature: Low Quality Consensus" twice in a row, then "Feature: Discrepancy".
Use the keyboard shortcuts to move forward and backward between features. You can also try changing the definition of features in the preferences, but perhaps wait until you read the next section.
Hopping from feature to feature in the contig view can be fun, but what if you want to get an overview of all features in a contig, and look at only some of the feature? You need the "Feature View" window! To see it, make sure that the contig view is the active window (click on it if in doubt), and then select "Features" from the "View" menu. This will open a new window that shows all the features in the contig in a table:
Now take a few seconds to rearrange your windows so that you can see both the contig view and the feature view. Then, double-click on a line in the feature view, for example the fourth line, which describes the discrepancy found in sample A455.s at base 103. Look at the contig view - the corresponding base in sample A455.s has been selected.
Double-clicking will also open the trace view window for this contig, and select the feature you clicked on in the trace view. You may need to re-arrange the windows so you can see the trace view as well as the contig and feature views.
Next, double-click on the feature view line that describes the "gap in contig" at base 186. You will see that this consensus base becomes selected in the contig view, and that two traces which have aligned bases at this position are added to the trace view window, with the bases at this feature selected:
Looking at the aligned traces makes it clear that the consensus sequence is correct.
You can also print and export the feature view, but that requires a trial license (or a purchased license), and is therefore described in the second part of this Quick Tour, together with all the other functions that require a trial or purchased license.
This section shows how to use tags for navigation. We use an example from the program PolyPhred, a widely used program for detecting heterozygous mutations in sequence traces. PolyPhred was developed by Dr. Deborah Nickerson and co-workers at the University of Washington; we are using it here as an example because PolyPhred uses tags to mark mutations
Even if you do not plan to use PolyPhred, we suggest you read on, since this section also introduces "tags". Tags can be added by programs by PolyPhred or by you to mark regions of interest in a sequence, and can be used to quickly find such regions.
To get started, open the PolyPhred example project that is included
in the Aligner example files. Close your current project if you still have
it open. Select "Open" from the "File" menu, and navigate to the example folder.
On OS X, the default location is:
/Applications/CodonCode Aligner/Example Files/PolyPhred_example. On Windows, the default location is:
C:\Program Files\CodonCode Aligner\Example Files\PolyPhred_example. Select the "PolyPhred_example.proj" file in this directory (depending on your system settings, you may not see the ".proj" extension). Aligner will read the project data and open a project window, which should show two contigs, one with 4 samples and the other with 6 samples.
To look at the tags added by PolyPhred, we first need to change our definition of what features are. Select "Define Features..." from the "Go" menu, and then uncheck all checkboxes. Click on the "Some tags" radio button to select it; your preferences should now look like this:
Now click on the "Specify..." button next to the "Some tags" radio button. This will open a dialog where you can select which tags are of interest to you which looks like this:
When you open the dialog, the tag selection is probably different. Make sure only Mutation tags are selected by first clicking on the "Select None" button, and then clicking on the "All Mutation tags" checkbox. Then, click "OK" in the tag selection dialog.
Before clicking "OK" in the preferences dialog, change one more setting: select the "Views" panel by clicking on the word "Views" on the left side of the preference dialog, and make sure that the check box in the middle to automatically pick traces is selected. Then, change the number of traces to pick automatically to 5, and now click "OK".
Now we are ready to look at the data. In the project view, double-click on "Contig1" to open the contig view. Also open the trace view and feature view for the contig (using the menu items or keyboard shortcuts). Now, double-click on a line in the feature view, for example the line that describes a heterozygous C-T at position 109 in sample CA-21.s. Your windows should look like this:
The tags PolyPhred added are color-coded in the contig view and trace view. Pink tags indicate a heterozygous base, blue tags a homozygous base. In this example, it is easy to see that PolyPhred classified all bases at this point correctly. You can quickly check the other mutation spots by going back to the feature view and double-clicking on another line, or by using the keyboard shortcuts to move to the next feature (Command-Right arrow on OS X, Control-Right arrow on Window).
Play around with the feature-navigation a bit, and then go on to the next chapter. We will explain how to export feature lists in the second part of the Quick Tour.
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