Sequencher Basics Tutorial
Loading Sequence Files
Begin by starting the Sequencher program. For this tutorial, we will be using some of the
sample files that came with the program. From the File menu select Import, and from
the resulting menu, click Sequences…
Navigate to the directory where you installed Sequencher. From this directory, navigate
to \Sample Data\T.G. Sample Data\ADARC. Select all of the files in this directory and
click Open.
You should now have the sequences ADARC1 through ADARC8 loaded into your
Sequencher Project window as pictured here:
Preparing the Sequences for Assembly
Before assembling the sequences, they must be trimmed to remove bad data from the
ends. For this example we will be using a trimming method based on the number of
ambiguous base calls. Sequencher also supports trimming based on quality scores and
vector contamination. For more information and examples of these methods, please see
the Trimming Tutorial included in the Sequencher installation.
To open the trimming window, select Trim Ends… from the Sequence menu.
Click on the Change Trim Criteria button at the top of the window. You should see the
following:
Check the boxes as pictured above. For the 5 prime end, change the numbers so that the
statement reads:
Trimming no more than 25%, trim until the first 50 bases contain less than 1 ambiguities.
For the 3 prime end, change the numbers so that the statement reads:
Starting 100 bases after 5’ trim, trim the first 50 bases containing more than 1
ambiguities.
Click OK.
Note: When trimming based on ambiguity, it is very important to be aware of the quality
of your data. For this example we are using very high quality data with few ambiguities,
therefore, we have set very stringent thresholds. For lower quality data the thresholds
should be lowered.
You should now see the following window:
This view gives you an idea of how your chosen trimming criteria affect your sequences.
Using the information displayed here, you can adjust the trim criteria to allow the longest
sequence with the fewest ambiguities. Click Trim Checked Items. You will receive the
following warning:
Click Trim to continue.
Assembling Your Sequences
Assembling sequences is incredibly easy with Sequencher. Start by selecting all of the
ADARC sequences:
Click the Assembly Parameters button. You will see the following window:
For this tutorial, we will simply use the default settings. In future projects, you can adjust
the minimum match percentage and minimum overlap settings to reflect the quality of
your data. Also, it is generally a good idea to always use the dirty data algorithm, as it
will produce better assemblies. To learn more about these settings, and the ReAligner
option, please see the Assembly Strategies Tutorial included with your Sequencher
installation.
To continue, click OK. You will be brought back to the main Sequencher project
window. To begin assembling your sequences, click Assemble Automatically. You
will receive the following confirmation window:
Click OK, and you will see that the sequence files in your project window have been
replaced by a single contig file, Contig[0001].
Editing Your Contig
You can begin editing by double-clicking the contig file, bringing up the following
window:
This window shows the position of each sequence in the assembly as well as the open
reading frames found on the contig. To see the base pair alignment, click the Bases
button:
To view the traces as well as the bases, click Show Chromatograms:
From this view you can scan the contig for ambiguous bases and determine the proper
base call by examinging the traces of each sequence. To begin this process, click on the
first base of the consensus sequence (the bottom line in the Contig[0001] window). You
can skip ahead to the next ambiguous base by pressing Ctrl-N. To change a base call,
simply type the proper letter while the base is selected. To change a base to a gap, type a
colon ( : ). Scan through the entire consensus sequence, editing it as necessary. When
you are finished, simply close the chromatogram and Contig[0001] windows.
Exporting Your Consensus Sequence
The final step is to export your consensus sequence for use outside of Sequencher. To do
this, highlight your contig, and from the Contig menu, select Create New Seq From
Consensus…
You will first receive the following warning:
Because we have just edited the contig’s bases and do not plan to further alter it, this
warning can be disregarded. Click Create New Sequence. You will now be prompted
to name the sequence. For this example, just leave the default name and click OK.
A window will not pop up displaying your sequence:
Close this window, and select the sequence in the Sequencher product window. From the
File menu, select Export and from the resulting menu, click on Sequences…
You will now see a standard save file dialogue. You can choose from many different
types of sequence files. Select the type, give the file a meaningful name, and click on
Save.
This concludes our Sequencher tutorial. If you have further questions about how to use
the software, consult the tutorial files included in the Sequencher installation, or contact
JP Jin (jjin@email.unc.edu).