21) How to do multiple sequence alignment using MUSCLE

MUSCLE is an alternative to ClustalX or W to construct a multiple sequence alignment (MSA). MUSCLE is particularly good at handling large number of sequences for MSA and produces better quality alignments when numerous short partial sequences are present in the input fasta file. Below are the steps to run MUSCLE alignment using the command prompt:

1. Create a new folder on your computer and download MUSCLE to this folder from

http://www.drive5.com/muscle/downloads3.6/muscle3.6_win32.zip

2. In the same folder paste a copy of your input sequences to be aligned.

3. Now, follow the instructions below to run MUSCLE using the command prompt (Start-> All Programs -> Accessories -> Command Prompt; note that the procedures below for the command prompt are all case-sensitive, so make sure you type carefully!)

4. Now, navigate the command prompt to the folder you just created for MUSCLE alignment. Here are some tips to help you move around in the command prompt:

- to go one folder level backward: cd..
- to list content of director/folder: dir
- to go one folder level forward: cd

5. Once you have navigated to the folder, type “muscle –in –out ” to perform the alignment.



6. Close the command prompt when the alignment is complete.

Note: The output file is in “afa” format, which is not suitable for subsequent analyses by other programs. Therefore, you should convert it to the popular “aln” format. To do this, load the output file into ClustalX and then click on File -> Save Sequence as -> under format, select “Clustal format” -> under “Save Sequence As” at the bottom, change the name of the file extension from “output_file.afa” to “output_file.aln” -> click on the OK button.


Content by: Sye Bee & Asif M. Khan
Posted by: Sye Bee
Edited by: Asif M. Khan