BIRCH

TUTORIAL: Introduction to Ugene


  February 26, 2018

It will probably be useful to view these videos before doing this tutorial.
UniproUGENE User Manual
BACHREST documentation: $doc/fsap/rest.txt

Overview: This tutorial introduces the basic features of the Ugene environment. Ugene is a diverse collection of bioinformatics tools run through a multi-panel graphical interface.

Topics:

1. Create a working directory and copy files

I can't repeat this often enough. ALWAYS create a new directory for each project.

cd tutorials
mkdir Ugene
cd Ugene

mkdir Intro
cd Intro
go into tutorials directory
create a directory called Ugene
go into the Ugene directory

create a subdirectory called Intro
go into the Intro directory

In your Ugene/Intro directory, type the following command, or paste it into your terminal window:

cp -p $birch/local/tutorials/Ugene/Intro/*.gen .

(It is critical that you include the space-dot at the end of this line. The dot is Unix shorthand for the current working directory.)

Verify that these files were copied successfully by typing

ls -l

-rw-rw-r-- 1 psgendb psgendb 24914 Mar 13 20:04 pBI121.gen
-rw-rw-r-- 1 psgendb psgendb  5405 Mar 15 17:59 pBS_SK-.gen
-rw-rw-r-- 1 psgendb psgendb  5120 Mar 15 17:59 pBS_SK+.gen

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'.



>>>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 go to the Directories section.

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. Click on OK to return to your UGENE session.




To get started, open the GenBank entry for the pBI121 cloning vector using File --> Open. For comparative purposes, also open the GenBank entry in a text editor.



The sequence is displayed in Ugene's Sequence View, which consists of four components: the Overview, the Zoom view, the Details view and the Annotation editor. You'll see that the features annotated in the GenBank entry are displayed in the Overview. If you mouse-over a feature, you can see additional information.

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.


All four of the components of the Sequence view are synchronized, such that any operation on a feature in one view is reflected in the other views. For example, the nptII gene encoding the neomycin phosphotransferase enzyme is shown by two arrows between 2k and 4k on the circle. The innermost arrow is for the gene, and the outermost arrow is for the protein coding sequence (CDS). If you click on the gene (inner arrow), it will be selected in all views.

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.



There is sometimes a trade-off between having several views and screen space. The green "Toggle views" button lets 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 at far right.


You can also add some space by clicking on the Show/Hide Restriction sites button, which is circled in the lower, center pane.





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


After you're done saving your file, quit Ugene.
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

In this section, we will try two examples of cloning restriction fragments into vectors.

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

As a simple example, we will simulate cloning the gene cassette containing the GUS coding sequence under the control of the 35S promoter. First, we need to find some restriction sites that flank the 35S-GUS gene. This shouldn't be too hard, since the gene was cloned into a multiple cloning site to begin with.

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.


The smaller fragment, going from 4955..7982, should be the 3 kb fragment that we want to clone into Bluescript. Next, we need to find the same sites in Bluescript, and digest that vector as well.

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.


As before, we need to digest pBS_SK- 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.

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.


At this point it is useful to have a look at the sequence file that was saved when we created the new construct, pBS_SK-GUS.gb. It is important to note that while this file superficially resembles a GenBank file, it does not adhere strictly to the official GenBank format. Most notable:
Because Unigene "GenBank" files do not adhere to the GenBank standard, not all programs will process these files correctly. That being said, most programs that read GenBank format will be able to at least read the sequences correctly.

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.

Verify the that the restriction sites, select pBS_SK-GUS and choose DNA/RNA --> BACHREST. Save the output to pBS_SK-GUS.bachrest. 

The first few lines of the output verify the sequence length is 5978, and the topology is circular.

Examples of information for two sets of digests is also shown.
-----------------------------------------------------------
                     BACHREST   Version 09/30/2012

pBS-SK-GUS          Topology: CIRCULAR  Length:     5978 bp
-----------------------------------------------------------
Search parameters:
   Recognition sequences between    6 and   21 bp
   Ends: 5' protruding, Blunt, 3' protruding
   Type: Symmetric, Asymmetric
   Minimum fragments:     0     Maximum fragments:  6000
   Maximum fragments to print:    30
-----------------------------------------------------------

                                         # of
Enzyme          Recognition Sequence     Sites     Sites   Frags   Begin     End
--------------------------------------------------------------------------------

AarI            CACCTGC(4/8)                 2
                                                    1494    5248    2224    1493
                                                    2224     730    1494    2223

AatII           GACGT^C                      0

Excerpts from the file verify that both EcoRI and HindIII cut this construct once each.

Note that the coordinates for these sites differ slightly from those listed in Ugene. The reason is that Ugene lists the position of a site at the beginning of the recognition sequence, whereas BACHREST lists the position of a site at the 5' end of the cut site, which is the top strand of the fragment to the right of the cut. For example, for the HindIII site

5'A^AGCTT3'

Ugene lists he position of the site as the first A, wheras BACHREST lists the site at the second A.








EcoRI           G^AATTC                      1

                                                    3029    5978    3029    3028




HindIII         A^AGCTT                      1
                                                    5975    5978    5975    5974