TUTORIAL: Introduction to Ugene

March 23, 2021

• UGENE podcast, episode #2: Working with a sequence: basic operations part 1
• UGENE podcast #12: Searching restriction enzymes cut sites with UGENE

• Working sequences in Ugene
• Finding and displaying restriction sites in a sequence
• Creating restriction fragments from sequences
• Simulating cloning steps in-silico to create a construct
• Verifying constructs by restriction digestion 

1. Create a working directory and copy files

2. Visualizing sequences and sequence features in the Ugene Sequence viewer.

Next, open the BIRCH launcher, from which we can run any program in the BIRCH system. One way to launch BIRCH is to type 'birch' at the command line. This will open the BIRCH launcher in your ugene directory.

Alternatively, click on the BIRCH icon on your desktop . The BIRCH launcher will appear. Launch Ugene from the Sequence menu:



Alternatively, Ugene can be launched from the command line by typing 'ugene -ui'.

>>>First time setup for Ugene<<< On multiuser Linux systems, Ugene has difficulty writing temporary files. This is a difficult to reproduce problem, and seems to only affect some percentage of users. Nonethless, it is probably best to set a workaround so that the problem isn't encountered. The first time you run Ugene, go to Settings --> Preferences. In the Application Settings window go to the Directories section.
Find the Temporary files line. By default, the Path for temporary files goes to /tmp, which is writeable by all users. Nonethless, sometimes, Ugene gives an error message saying that files can't be written to /tmp. The workaround is as follows: Click on the "..." button to open a file chooser. In the file chooser, click on the name of your $HOME directory in the left column (shown as "psgendb" in this example). Next, go into the file pane and right click to bring up the menu. Choose "Show hidden files". This will show you directories whose names begin with ".". These directories are contain configuration files and other application-specific data.
Choose ".UGENE_files" directory and click on Choose. The Path for temporary files should now be set to this directory. Also, check the Documents line. If the Documents directory is not set to your home directory, change this line to point to your home directory, NOT, the $HOME/Documents directory. Click on OK to return to your UGENE session.

For example, there are two replication orgins in pBI121. the OriV origin is needed for this pBI121 to replicate in Agrobacterium tumefaciens.
The ColE1 origin is needed for replication in E. coli.

Note that the nptII gene feature is shown in the Overview and the Details view respectively as a box and an arrow. In the Details view, the coding sequence and the translation are both highlighted. In the Annotation editor, the annotation list has opened to the gene feature for nptII.

THESE BUTTONS WILL MAKE UGENE MUCH EASIER TO USE! There is sometimes a trade-off between having several views and screen space. Show/Hide buttons (in red dotted lines above) let you control which views are open.  For example, to make the Overview bigger, try using this menu to hide the Details view and the Zoom view. Next, you can make a bit more space for the Annotations editor by dragging the double line above the Annotations editor upwards. The result should look something like that shown in the next panel at far right.

You can also add some space by clicking on the Show/Hide Restriction sites button, which is circled at left.

Saving files as Ugene Projects

Ugene can organize sets of related files into projects. The contents of the current project are displayed in the Project bar at the left of the Ugene window. This would be a good time to save our work so far as a Ugene project. Choose File --> Save project as. Set Project name: UgeneIntro Project folder: choose the intro folder Project file: UgeneIntro

To demonstrate how to read a project back in, start Ugene again. Normally, Ugene will not load the project files until you explicitly ask them to be loaded. In this project, pBI121.gen is shown at right as [unloaded]. To load this file, select pBI121.gen and right click to open the load menu. Choose "Load selected document". At this point, Ugene will display pBI121 as shown previously.

3. Creating sequence constructs by simulated cloning

Cloning the 35S-GUS cassette from pBI121 to pBluescript SK(-)

Open the Analyze menu, which can be found in the Actions menu, or by right-clicking within the Overview pane. Choose Find Restriction sites, and then Open enzymes. Select compro.bairoch and click on Open. This file contains a subset of restrictions from the REBASE database, including only those enzymes commercially available, and only one enzyme (ie. the prototype) for each recognition sequence. By eliminating redundant enzymes, the display won't be cluttered.

Click on Select All to select all enzymes. Since we're only interested in enzymes that cut a maximum of twice in the plasmid, make sure that Minimum hits is set to 1, and Maximum hits is set to 2.  Click on OK to begin the search.

The restriction sites found will appear on the map, along with the annotations. Two promising sites are the HindIII site at around 5 kb, and the EcoRI site at around 8 kb.

If you open the Details view you can see the HindIII site by clicking on "HindIII" in the Overview. This will cause the Details view to jump to the HindIII site, AAGCTT. The cutting sites on both strands are indicated by small triangles superimposed over the sequence.  More complete information can be found by mousing over the HindIII site.

Next, we need to digest pBI121 with EcoRI and HindIII. Either go to the Actions menu at top, or right click in the Overview window and choose Cloning --> Digest into Fragments. Use the Add button to add EcoRI and HindIII to the Selected enzymes pane.  Click on OK.

To make some space, Hide the Details pane, and go to the Annotation Editor. If you open the tabs below "AF485783 features", you will see two fragments listed. Mousing over the fragments will show you details of these two EcoRI/HindIII fragments. Each fragment has one EcoRI end, and one HindIII end.

Choose File --> Open, and open the file pBS_SK-.gen. An entry for pBS_SK-.gen will appear in the project menu as shown below. At right, we can see the restriction map for Bluescript. Although the GenBank entry contains no annotation to be displayed, Ugene has thoughtfully done an automatic search for the same list of Restriction Enzymes. It is easy to see the multiple cloning site, centered around 700 on the circle.

The cloning operation is done as follows: Open the Actions menu, or right click in the Overview and choose Cloning --> Construct molecule. The fragments we want are the 3 kb fragment from pBI121, and the larger fragment, Fragment 1, from pBS_SK-. (The smaller fragment is a tiny 8 bp fragment between the HindIII and EcoRI sites.) Select each fragment you want and use the Add button to add them to the new construct. Also, make sure that the "Annotate fragments" and "Make circular" boxes are checked. The ends of the two fragments are shown in green. Next, go to the Output tab, and set the output file name to pBS_SK-GUS.gb. Click on OK to generate the new construct.

The new construct will appear in a new Ugene tab. Initially, Ugene will display all of the restriction sites in the Overview. To make the view a bit less cluttered, redo the restriction search from before using Analyze --> Find Restriction Sites. However, in the Find Restriction Sites window, first click on the Select None button. Next, open the +E and +H tabs and check ONLY EcoRI and HindIII. Click on OK to display the new view.

You can save maps from Eugene at any time as a bitmap graphic file. Right-click in the Overview window and choose Export --> Save circular view as image. For this example, call the file pBS_SK-GUSmap.png. Ugene saves files in PNG format by default, but a variety of formats are supported. You can view the file by opening it from the file manager, or importing it into a document.

To make it absolutely clear what is happening in the cloning process, the diagram at right shows how pBI121 Fragment2  and pBS_SK- Fragment 1 ligate together.

• On the LOCUS line, the word 'circular' should be in columns 56-63. Ugene writes this word in column 49-56.
• There is no DEFINITION section
• The UNIMARK line is not part of the GenBank format
  
• There is no ACCESSION number. (This is in part because only NCBI can assign ACCESSION numbers.)
• many of the qualifier lines in the FEATURES table are Ugene-specific eg. "ugene_name".
  

To launch bldna, either type 'bldna' at the command line (make sure you are in the intro directory first), or from the BIRCH launcher, choose Sequence --> bldna, and set the working directory to the intro directory. Choose File --> Open and pBS_SK-GUS.gb.