Koichiro Tamura, Daniel Peterson, Nicholas Peterson, Glen Stecher, Masatoshi Nei, and Sudhir Kumar (2011) MEGA5: Molecular Evolutionary Genetics Analysis using Likelihood, Distance, and Parsimony methods. Molecular Biology and Evolution.
(to be submitted)
Kumar S, Dudley J, Nei M & Tamura K (2008) MEGA: A biologist-centric software for evolutionary analysis of DNA and protein sequences. Briefings in Bioinformatics 9: 299-306.
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Tamura K, Dudley J, Nei M & Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24: 1596-1599.
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Creating Multiple Sequence Alignments

In this example, we will create an alignment from protein sequence data that will be imported into the alignment editor using different methods.

Ex 1.0.1: Start MEGA by double-clicking on the MEGA desktop icon, or by using the Windows start-menu to click on the MEGA icon located in the programs folder.

Ex 1.0.2: Launch the Alignment Explorer by selecting the Alignment|Alignment Explorer/CLUSTAL menu command.

In order to align sequences contained in a Sequence Data File, do the following:

Ex 1.1.1: Add unaligned sequences from the hsp20.fas example file into the Alignment Explorer by clicking selecting the Data|Open|Retrieve Sequences from File menu command.

Ex 1.1.2: Select the Edit|Select All menu command to select every site for all sequences in the alignment.

Ex 1.1.3: Select the Alignment|Align by ClustalW menu command to align the selected sequences data using the ClustalW algorithm.

Ex 1.1.4: Save the current alignment session by selecting the Data|Save Session menu item. This will allow the current alignment session to be restored for future editing.

Ex 1.1.5: Exit the Alignment Explorer by selecting the Data|Exit Alignment Explorer menu item. A message will appear asking if you would like to save the data to a MEGA file. Choose “YES,” and then a “Save As” dialog box will appear. Enter hsp20_aligned.meg as the file name, and click the “Save” button. An input box will appear asking for a title for the data. Enter “HSP 20 Aligned by MEGA” as the title, and click the “OK” button. Another dialog box will appear asking you if the sequence data is protein coding. In this case, click “Yes.” A final dialog box will appear asking you if you would like to open the data file in MEGA. Click “Yes.”

Now, we will examine how to send sequence data from the Internet (Web Explorer) to the Alignment Explorer.

Ex 1.2.1: If the Alignment Explorer already contains sequence data, select the Alignment | Create a new alignment menu command to create a new alignment. Choose “YES” on the dialog box that appears to indicate that you are creating a DNA sequence.

Ex 1.2.2: Activate the Web Explorer tab by selecting Web|Query Gene Banks from the menu.

Ex 1.2.3: When the NCBI Entrez site is loaded, select either the nucleotide or protein database, enter a search term into the search box, and press the “GO” button.

Ex 1.2.4: When the search results are displayed, select the specific search item and choose “Sequence” from the menu bar. Press the “Add to Alignment” button located to the left of the address box. This will display the Web Fetch dialog window.

Ex 1.2.5: Click the box to the left of each accession number whose sequences’ information you would like to fetch from the web. When you are done, select accessions by pressing the “Fetch” button.

Ex 1.2.6: When the status column indicates that all sequences are fetched, press the “Send to Alignment” button to send the fetched sequence data to the Alignment Explorer.

Ex 1.2.7: Align the fetched data using the steps detailed in Ex 1.1.2 – Ex 1.1.5.

You may also open a trace file in the Trace Data Viewer/Editor and send it directly to the Alignment Explorer.