The basis of assembly is similar to the multiple sequence alignments that we have looked at. In essence we are looking for regions of homology at the ends of the DNA sequence. We will then try and align these sequences together to create a single sequence like this:
Of course, each sequence could be homologous at either end to another sequence, and each sequence could be homologous on either strand at either end to another sequence, and so the possibilities get daunting if you were to do this by hand. Luckily Sequencher has built in functions to do this. Go to the main sequencher window:
Now take a look at the top line below the buttons. Notice that it says:
These are the current assembly parameters, but we can change these. Click on the Assembly Parameters button. That will pull up this window:
There is a text box that decides the overall parameters for clean data and dirty data, and sliders where you can change the minimum match percentage and the minimum overlap. For many (most) alignments the defaults will be satisfactory, but you may want to alter these values particularly if your data is poor at the end of a sequence and may not match up. Click OK to accept these values and we will continue with the alignment.
When you click OK you are returned to the main window. At the moment we will only consider automatic assembly. It is generally sufficient for most assemblies, but you can assemble sequences manually if you wish, and these will be covered later.
Click the Assemble Automatically button. If you do not have any sequences highlighted, it will warn you that there is an error, and that you need to highlight some sequences. Highlight them all and try again.
The assembly will be rapid and take about 3 or 4 passes through the data. At each pass it makes an alignment and then on the next pass it compares that alignment to the other alignments and the other sequences. The more sequences you have, the more alignments it will make and the more passes it will need. Notice that the 
icon has changed to 
to indicate that this is a contig. Also notice that the size has increased larger than any of the single sequences. This is the size of the contig. Finally notice that the kind says "Contig of x sequences". Click on the little triangle next to the contig icon and the contig will expand to list all the sequences inside of it like this:
Now double click on the contig name. You will see a new window open with a graphical view of the contig:
There are many things to note about this view:
No coverage in the contig.
One strand sequenced once.
One strand sequenced more than once.
Both strands sequenced once.
Both strands sequenced, some more than once.
) and stop codons are shown as red lines (
).
There are a large number of options from this view, and we will explore some of them below. Start by clicking on the bases window. You will see something like this:
(Note that I scrolled along the window some.) Notice that the sequence is given, and that secondary base calls are highlighted in pink as we have seen before. Mismatches are denoted by a 
and conservative mismatches are denoted by a + sign.
Highlight a selection of bases by clicking on the consensus sequence. All bases should highlight as shown in the above image. Now click the Show Chromatograms button. You will see a view like this:
There are several things to notice in this view:
becomes a 
and so on.
to 