Copyright Jon Monroe and Louise Temple, 3/8/07

In this exercise, you will see how to edit raw DNA sequence data, create an overlapping “contig”, determine open reading frames, and check for homology to known genes/proteins.  The data you will use represent three sequencing reads from a bacteriophage genome used in the Genomics class, spring 2006.

1.  Launch the program Sequencher 4.6 Win Demo from the desktop.  Acknowledge that this is a Demo version. 

2.  Click on File and select Import, then Folder of Sequences.  Find the CURData folder and press OK.

3.  Three files will be loaded into Sequencher; data1.ab1, data2.ab1 and data3.ab1.  All three will be highlighted by default so to open only the first file, click in the window to deselect the files then double click on the data1.ab1.  A new window with the sequence will appear.

4.  Spend a few moments to become familiar with the program views and menus.  You can toggle between a "Bases" view (the default) and an "Overview" illustrating various aspects of the sequence.  In the bases view click on Cut Map to see the restriction sites in this sequence.  Notice the clustered sites near the 5' end indicative of a multiple cloning site.  This part of the sequence will be trimmed below.

5.  Now click on Bases to return to the previous window, then click on Overview.   The Diagram Key will be helpful here but notice that the sequence is illustrated in three reading frames.  Since stop codons are illustrated by red hatch marks, which reading frame is likely to contain a gene sequence?

6.  Now return to the bases view and choose Show Chromatogram to view the raw data from the sequencing machine.  Find the many “Ns” at the 3' end of the sequence.  N is the single-letter code for any base indicating that the sequence was ambiguous in this region.  These will also be trimmed below.  About how many good bases are present in this sequence?

7.  Look for the cloning site at about base #100; these clones were partial digests using Tsp509I, a 4-base cutter with ends compatible with EcoRI.  In some, but not all, cases, the EcoRI site is retained (GAATTC).  Before proceding to the next section, close the window containing the sequence.

Now you are ready to do some editing.  We recommend that the students first learn how this is done by looking for the cloning sites on both ends of some sequences and then trimming them manually.  Later, when lots of sequences are being processed they will really appreciate the automated process...

8.  Under Select, choose Select All, then Sequence, then Trim Vector....  This will pull up a dialog box.

9.  Select Choose Insertion Site Now, then click the first UseVecbase File button (to trim one end of each sequence).  Choose the workshop CD then select 3 Vectors from VecBase.

10.  Choose BlueKSm then click the Select button.

11.  Click and highlight the EcoRI site and hit OK.

12.  To trim the other end of each sequence, repeat this procedure by clicking the Use Vecbase file in the second window, select  BlueKSm then the EcoRI site, but this time press Prime Other Strand then OK.  This permits trimming from both directions in the vector.  Close out of the window.

13.  Pull down Sequence and select Trim Vector...  Note: you should see scissors illustrating cutting from each end of two of the three sequences (only those sequences with detectable vector appear in the box).   Select Trim Checked Items and proceed in spite of the caution box.  Note the change in length of each sequence, which is also reflected in the original window.  Close the window and repeat this process until you see a dialog box indicating that no vector contamination was found.

14.  Once that is finished close the window and under the sequence menu click Trim Ends in order to remove the ambiguous bases that are present at the end of some sequences (highlighted in red).  Now click Trim Checked Items then close the window.

Look at the assembly parameters.  Using this dialog box, you can change the stringency of matches and work with the data in different ways.  We will use the default, which is “Dirty Data” with 85% minimum match and 20 bases minimum overlap.  Now close that window.

15.  Click the Assemble Automatically button on the main window.  This will begin assembling forward and backwards strands to form contigs.  The results are shown in a box which you can close by clicking Close.  One contig should be formed and zero fragments should remain.

16.  Open the contig by double-clicking on it.  The resulting box should look like the single sequence you were viewing earlier, except that it has the single sequences above the contig.  You can look at the bases, or an overview, and at the chromatogram from this box.

17.  Choose the bases option and open the box wider.  Note that  the consensus sequence is at the bottom.  Scroll to the right to see a region of overlapping sequences, click on the consensus sequence line where there are overlapping sequences (scroll to the right), then choose Show Chromatogram. This will allow you to view the raw data to resolve sequence ambiguities.

18.  Scroll along the sequence until you find an ambiguous base.  Click on the ambiguous base in the consensus sequence, and the same base will be highlighted in each chromatogram.  You can choose to change the consensus line based on the chromatograms.  Where 2 of the 3 bases are the same the consensus will likely be correct but where there are only two sequences and they differ an ambiguity code will occur in the consensus line.  Through this process students should easily learn the value of having the same region sequenced many times to increase the level of confidence in the sequence...

19.  Go back to the Overview and inspect the clones.  How much overlap is there in this contig?  Are both strands sequenced multiple times in both directions?  What would it take to get “8-fold coverage” as advertised in many sequencing projects?