Introduction
MUSCLE stands for MUltiple Sequence Comparison by Log-Expectation. MUSCLE is claimed to achieve both better average accuracy and better speed than ClustalW2 or T-Coffee, depending on the chosen options. MUSCLE enables high-throughput applications to achieve average accuracy comparable to the most accurate tools previously available, which is expected to be increasingly important in view of the continuing rapid growth in sequence data. Multiple alignments of protein sequences are important in many applications, including phylogenetic tree estimation, secondary structure prediction and critical residue identification.
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Important Note
- This is NOT a pairwise alignment tool. To align two sequences please select a service from the pairwise alignment tools section.
- MSA tool algorithms are NOT intended to produce genome synteny maps.
- There is currently a limit of 500 sequences or a maximum file size of 1MB of data.
- We kindly ask all users of EMBL-EBI Web Services to submit tool jobs in batches of no more than 30 at a time and not to submit more until the results and processing is complete. Please ensure that a valid email address is provided. Excessive usage of a particular resource will be dealt with in accordance with EMBL-EBI's Terms of Use. Please contact us if you need further information.
How to Access MUSCLE
You can access the tool via
Web Form
Web interface for Muscle is available at : https://www.ebi.ac.uk/Tools/msa/muscle/
How to use this tool
Running a tool from the web form is a simple multiple steps process, starting at the top of the page and following the steps to the bottom.
Each tool has at least 2 steps, but most of them have more:
- The first steps are usually where the user sets the tool input (e.g. sequences, databases...)
- In the following steps, the user has the possibility to change the default tool parameters
- And finally, the last step is always the tool submission step, where the user can specify a title to be associated with the results and an email address for email notification. Using the submit button will effectively submit the information specified previously in the form to launch the tool on the server
Note that the parameters are validated prior to launching the tool on the server and in the event of a missing or wrong combination of parameters, the user will be notified directly in the form.
Step 1 - Sequence
Sequence Input Window
Three or more sequences to be aligned can be entered directly into this form. Sequences can be in GCG, FASTA, EMBL, GenBank, PIR, NBRF, PHYLIP or UniProtKB/Swiss-Prot format. (See example input formats). Partially formatted sequences are not accepted. Adding a return to the end of the sequence may help certain applications understand the input. Note that directly using data from word processors may yield unpredictable results as hidden/control characters may be present.
Sequence File Upload
A file containing three or more valid sequences in any format (GCG, FASTA, EMBL, GenBank, PIR, NBRF, PHYLIP or UniProtKB/Swiss-Prot) can be uploaded and used as input for the multiple sequence alignment. (See example input formats). Word processor files may yield unpredictable results as hidden/control characters may be present in the files. It is best to save files with the Unix format option to avoid hidden Windows characters.
STEP 2 - Set your Parameters
Output Format
Format for generated multiple sequence alignment. (See example output formats).
| Output Format | Description | Abbreviation |
|---|---|---|
| Pearson/FASTA | Pearson or FASTA sequence format | fasta |
| ClustalW | ClustalW alignment format without base/residue numbering | clw |
| ClustalW (strict) | Strict ClustalW alignment format without base/residue numbering | clwstrict |
| HTML | HTML format colored alignment | html |
| GCG MSF | GCG Multiple Sequence File (MSF) alignment format | msf |
| Phylip interleaved | PHYLIP interleaved alignment format | phyi |
| Phylip sequential | PHYLIP sequential alignment format | phys |
Default value is: ClustalW [clw]
Output Tree
The guide tree to output
| Matrix (Protein Only) | Abbreviation |
|---|---|
| none | none |
| From first iteration | tree1 |
| From second iteration | tree2 |
Default value is: none
Step 3 - Submission
Job title
It's possible to identify the tool result by giving it a name. This name will be associated to the results and might appear in some of the graphical representations of the results.
Email Notification
Running a tool is usually an interactive process, the results are delivered directly to the browser when they become available. Depending on the tool and its input parameters, this may take quite a long time. It's possible to be notified by email when the job is finished by simply ticking the box "Be notified by email". An email with a link to the results will be sent to the email address specified in the corresponding text box. Email notifications require valid email addresses.
Email Address
If email notification is requested, then a valid Internet email address in the form joe@example.org must be provided. This is not required when running the tool interactively (The results will be delivered to the browser window when they are ready).
Web Services
Web Services are available using REST and SOAP protocols that enable programmatic access and allow their integration into other applications and analytical workflows and pipelines.
For an introduction on how to run these clients and use them in workflows please see the webinar series.
REST API
The Representational State Transfer (REST) sample clients are provided for a number of programming languages. For details of how to use these clients, download the client and run the program without any arguments.
Language | Download | Requirements |
|---|---|---|
| Perl | muscle.pl | LWP and XML::Simple |
Python | xmltramp2 |
For details see Environment setup for REST Web Services and Examples for Perl REST Web Services Clients pages.
WADL
The WADL for the MUSCLE (REST) service: http://www.ebi.ac.uk/Tools/services/rest/muscle?wadl
Tool Parameters
Parameters | Command line parameter |
|---|---|
OUTPUT TREE | --tree |
| OUTPUT ORDER | --order |
SOAP API
The Simple Object Access Protocol (SOAP) sample clients are provided for a number of programming languages. For details of how to use these clients, download the client and run the program without any arguments.
WSDL
The WSDL for the MUSCLE (SOAP) service: http://www.ebi.ac.uk/Tools/services/soap/muscle?wsdl
The Kissinger Research Group, University of Georgia provide a SAWSDL for the MUSCLE (SOAP) web service, which is annotated using OBIws: http://mango.ctegd.uga.edu/jkissingLab/SWS/Wsannotation/resources/muscle.sawsdl
Open API Interface
https://www.ebi.ac.uk/Tools/common/tools/help/index.html?tool=muscle
Common Workflow Language
CWL (Common Workflow Language) implementation for consuming EMBL-EBI Bioinformatics Web Services tools' clients are available at https://github.com/ebi-wp/webservice-cwl
For details, see CWL Workflows page.