Final exam - full solution

Full solution.

The DNA sequence in our new allele (isolate 80378) is correctly aligned from nucleotide site 179 (amino acid site 60) onwards.

The translated protein sequence should actually begin at site 26 in our new allele sequence. (Hint: type ‘26’ in the ‘Site #’ box at the bottom left of the alignment window in MEGA if you need help to find it).

It looks as though you could (i) remove superfluous sequence at the beginning of our new allele, (ii) move the section of sequence in the black box to match the beginning of the other alleles CAMP0073 1-8 and (iii) we would then need to create a gap between the moved sequence within the box, and the portion of sequence that is already correctly aligned.

Alleles and isolate sequence open in MEGA. Shown with colour. Arrows to indicate where the sequence is the same and where a deletion has occurred.
Alleles and isolate sequence open in MEGA. Shown without colour. Arrows to indicate where the sequence is the same and where a deletion has occurred.

You can then test if your theory is correct by manipulating your new sequence to align with existing alleles. You can do this by returning to the DNA sequences alignment view in MEGA.

Then, you can (i) delete the superfluous nucleotides before the correct starting point of your sequence using the backspace button; and (ii) add spaces using the space bar. If you have made a mistake, you can use the Edit tab and Undo option, or just type the letters that you need.

You should end up with something like this, where you can visualise the point where the deletion has happened.

Alleles and isolate sequence open in MEGA. Shown with colour. The deletion portion is indicated.
Alleles and isolate sequence open in MEGA. Shown without colour. The deletion area is shown.

It is advisable to give one last check to the translated protein sequence to ensure you haven’t introduced internal stop codons etc.

Alleles and isolate amino acid sequence open in MEGA. Shown with colour. Arrows to indicate where the sequence is the same and where the deletion has occurred.
Alleles and isolate amino acid sequence open in MEGA. Shown without colour. Arrows to indicate where the sequence is the same and where the deletion has occurred.

Super advanced level!

One final thing.  The super curious and observant among you may have noticed that you can use automated sequence alignment tools called ‘ClustalW’ and ‘MUSCLE’ in MEGA.  To use them, select your new (unaligned) sequence and at least one other sequence in the DNA sequence alignment by holding down the shift button; then select the alignment tool you would like to use. Use the ‘Help’ tab on MEGA if you would like more information about each of the options.

Check to see what happens to your new sequence.  Do you agree with the sequence alignment created by the automated tool(s)?