TUTORIAL: Introduction to Ugene
February 26, 2018
|go into tutorials directory
create a directory called Ugene
go into the Ugene directory
create a subdirectory called Intro
go into the Intro directory
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.
example, there are two replication orgins in pBI121.
the OriV origin is needed for this pBI121 to replicate in Agrobacterium tumefaciens.
ColE1 origin is needed for replication in E. coli.
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.
sometimes a trade-off between having several
views and screen space. The green "Toggle
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.
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.
Project name: UgeneIntro
Project folder: choose the Intro folder
Project file: UgeneIntro
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
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.
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
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.
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
restriction sites found will appear on the map, along with
Two promising sites are the HindIII site at around 5 kb, and the EcoRI site at around 8 kb.
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
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.
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.
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.
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
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.
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
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.
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.
|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.|
|The first few lines of the output
verify the sequence length is 5978, and the topology is
Examples of information for two sets of digests is also shown.
BACHREST Version 09/30/2012
pBS-SK-GUS Topology: CIRCULAR Length: 5978 bp
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
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
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