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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. 

Official Website

Download Software

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 FormatDescriptionAbbreviation
Pearson/FASTAPearson or FASTA sequence formatfasta
ClustalWClustalW alignment format without base/residue numberingclw
ClustalW (strict)Strict ClustalW alignment format without base/residue numberingclwstrict
HTMLHTML format colored alignmenthtml
GCG MSFGCG Multiple Sequence File (MSF) alignment formatmsf
Phylip interleavedPHYLIP interleaved alignment formatphyi
Phylip sequentialPHYLIP sequential alignment formatphys

Default value is: ClustalW [clw]

Output Tree

The guide tree to output

Matrix (Protein Only)Abbreviation
nonenone
From first iterationtree1
From second iterationtree2

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).

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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

Perlmuscle.plLWP and XML::Simple

Python

muscle.py

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 

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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 OBIwshttp://mango.ctegd.uga.edu/jkissingLab/SWS/Wsannotation/resources/muscle.sawsdl


 List Methods
MethodsDescriptionArgumentsReturns

getParameters()

Get a list of the parameter namesnonea list of strings giving the names of the parameters.

getParameterDetails(parameterId)

Get details of a specific parameterparameterId: identifier/name of the parameter to fetch details ofa wsParameterDetails describing the parameter and its values.

run(email, title, params)

Submit a job to the service

email: (required) user e-mail address. See Why do you need my e-mail address?

title: job title. Default: ””

params: (required) parameters for the tool. These are described by the InputParameters data structure.

a string containing the job identifier (jobId).

getStatus(jobId)

Get the status of a submitted job.jobId: (required) job identifier.

a string containing the status.

The values for the status are:


RUNNING: the job is currently being processed.

FINISHED: job has finished, and the results can then be retrieved.

ERROR: an error occurred attempting to get the job status.

FAILURE: the job failed.

NOT_FOUND: the job cannot be found.

Further details can be found in Synchronous and Asynchronous Access: Job Dispatcher.

getResultTypes(jobId)

Get the available result types for a finished jobjobId: (required) job identifier.a list of wsResultType data structures describing the available result types.

getResult(jobId, type, parameters)

Get the result of a job of the specified type.

jobId: (required) job identifier.

type: (required) string specifying the result type to fetch. See getResultTypes(jobId) for details of the available types.

parameters: optional list of wsRawOutputParameter used to provide additional parameters for derived result types.

the result data for the specified type, base64 encoded. Depending on the SOAP library and programming language used the result may be returned in decoded form. For some result types (e.g. images) this will be binary data rather than a text string.



 List Datatypes

InputParameters

The input parameters for the job:

AttributeTypeDescription
formatstringalignment format
treestringtree type
orderstringorder of sequences in the alignment
sequencestringsequences to align

More detailed information about each parameter, including valid values can be obtained using the getParameterDetails(parameterId) operation.

wsParameterDetails

Descriptive information about a tool parameter. Returned by getParameterDetails(parameterId).

AttributeTypeDescription
namestringName of the parameter.
descriptionstringDescription of the parameter, suitable for use in option help interfaces.
typestringData type of the parameter.
valueslist of wsParameterValueList of valid values for the option.

wsParameterValue

Description of a tool parameter value. Used in wsParameterDetails.

AttributeTypeDescription
labelstringDisplay name of the value, for use in interfaces.
valuestringString representation of the value to be passed to the tool parameter.
defaultValuebooleanFlag indicating if this value is the default.
propertieslist of wsPropertyList of key/value pairs providing further information.

wsProperty

Properties of a tool parameter value. Used in wsParameterValue.

AttributeTypeDescription
keystring
valuestring

wsRawOutputParameter

Additional parameters passed when requesting a result. See getResult(jobId, type, parameters).

AttributeTypeDescription
namestring
valuelist of string

wsResultType

Description of a result type. Returned by getResultTypes(jobId).

AttributeTypeDescription
identifierstringIdentifier for the result type. Passed as type to getResult(jobId, type, parameters).
labelstringDisplay name for use in user interfaces.
descriptionstringDescription of the result type, for use in help interfaces.
mediaTypestringMIME type of the returned data.
fileSuffixstringSuggested suffix for file name, if writing data to disk

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Open API Interface

https://www.ebi.ac.uk/Tools/common/tools/help/index.html?tool=muscle


 List Endpoints

Endpoints

Description

Arguments

Returns

https://www.ebi.ac.uk/Tools/services/rest/muscle/parameters

Get a list of the parameter names.noneA list of strings giving the names of the parameters.

https://www.ebi.ac.uk/Tools/services/rest/muscle/parameterdetails/format

https://www.ebi.ac.uk/Tools/services/rest/muscle/parameterdetails/tree

https://www.ebi.ac.uk/Tools/services/rest/muscle/parameterdetails/sequence

Get details of a specific parameter.parameterdetail: identifier/name of the parameter to fetch details of.A wsParameterDetails describing the parameter and its values.
https://www.ebi.ac.uk/Tools/services/rest/muscle/run
Submit a job to the service.

email: (required) user e-mail address. See Why do you need my e-mail address?

title: job title. Default: ””.

params: (required) parameters for the tool. These are described by the InputParameters data structure.

A string containing the job identifier (jobId).
https://www.ebi.ac.uk/Tools/services/rest/muscle/status/<jobid>
Get the status of a submitted job.jobId: (required) job identifier.

A string containing the status.

The values for the status are:

  • RUNNING: the job is currently being processed.

  • FINISHED: job has finished, and the results can then be retrieved.

  • ERROR: an error occurred attempting to get the job status.

  • FAILURE: the job failed.

  • NOT_FOUND: the job cannot be found.

Further details can be found in Synchronous and Asynchronous Access: Job Dispatcher.

https://www.ebi.ac.uk/Tools/services/rest/muscle/resulttypes/<jobid>

Get the available result types for a finished job.

jobId: (required) job identifier.

Returns: a list of wsResultType data structures describing the available result types.

https://www.ebi.ac.uk/Tools/services/rest/muscle/result/<jobid>/out

https://www.ebi.ac.uk/Tools/services/rest/muscle/result/<jobid>/error

https://www.ebi.ac.uk/Tools/services/rest/muscle/result/<jobid>/sequence

https://www.ebi.ac.uk/Tools/services/rest/muscle/result/<jobid>/phylotree

https://www.ebi.ac.uk/Tools/services/rest/muscle/result/<jobid>/pim

Get the result of a job of the specified type.

jobId: (required) job identifier.

type: (required) string specifying the result type to fetch. See getResultTypes(jobId) for details of the available types.

parameters: optional list of wsRawOutputParameter used to provide additional parameters for derived result types.

Returns: the result data for the specified type, base64 encoded. Depending on the SOAP library and programming language used the result may be returned in decoded form. For some result types (e.g. images) this will be binary data rather than a text string.

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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. 

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Reference 

MUSCLE

EBI  Web Services

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