Opening Allele Sequences in MEGA

MEGA is a piece of software to view and align sequences. You get a visual understanding of the large number of defined alleles and their differences. We will use this software to compare our isolate sequence to the defined alleles and see how our isolate sequence is unique. 

Download here from MEGA's website.               

Using MEGA to look at our isolate sequence:

1. Open the program and select "ALIGN" in the top left

2. From the drop-down, select the first option "Edit/Build Alignment"

3. Select "Retrieve a sequence from a file" and then "OK". This will open a new window and file explorer. 

4. Select the file containing the allele sequence (download previously from PubMLST). In the example from the previous guide NEIS0051.

5. The result will be like the image below, which has arrows to allele numbers, site in sequence and amino acid conversion tab.

The column on the left extending down the page (allele numbers) is the name of the gene and the number that allele has been given. Going horizontally across is the sequence for all the alleles. 

The "Translated Protein Sequences" tab gives the option to translate the sequence - see it as its amino acids. Select “Yes” to “The current Genetic Code is: Standard. Is this correct?”.

The box in the bottom left, shows you the site of the sequence you are at if you click on a base (or amino acid) but also can be used to find a certain base. 

1. Select a base (A/T/G/C) and the box will display its place in the sequence.
2. Select any base in a row and then type in a number to see the base at that place in the sequence.

6. To insert our sequence, click on the DNA sequences tab, then select Edit

7. Then select "Insert Blank Sequence" from the drop-down

8. Then make sure you only have one box selected, it should look as below - one single grey box with the rest of the row in white. If it doesn't and is all grey, click on the first box again.

9. You can then paste in your sequence - shown is a short section ATGCGTCAGTAGGGA. Feel free to try with this sequence - simply copy the text but do not include the full stop. It should look like this:

10. You can rename the sequence by right clicking the name and selecting edit sequence name. You can also re-arrange the sequences by dragging the names, use this to drag our sequence to the top.

In this example, the alleles are identical for the first part. If you use scroll to the right using the bar on the bottom you can see that the further you go, the more differences there are in sequences.

Walkthrough video:

The video discusses opening the alleles for CAMP0021, from Campylobacter jejuni/coli. The written walkthrough above is for NEIS0051, from Neisseria ssp. The process is the same for both.