View source for Clustal

{{Short description|Bioinformatics computer program}}
{{Tone|date=April 2022}}
{{Infobox software
|name                   = CLUSTAL
|developer              = {{Plainlist|
* [[Desmond G. Higgins|Des Higgins]]
* Fabian Sievers
* David Dineen
*  Andreas Wilm (all at the Conway Institute, [[University College Dublin|UCD]])}}
|latest_release_version = 1.2.2
|latest release date    = {{release date and age|2016|07|01|df=yes}}
|operating_system       = [[UNIX]], [[Linux]], [[Apple Macintosh|MacOS]], [[MS-Windows]], [[FreeBSD]], [[Debian]]
|programming language   = C++
|genre                  = Bioinformatics tool
|licence                = [[GNU General Public License]], version 2<ref>See file COPYING, in source archive [http://www.clustal.org/omega/clustal-omega-1.2.0.tar.gz] {{Webarchive|url=https://web.archive.org/web/20210612074734/http://www.clustal.org/omega/clustal-omega-1.2.0.tar.gz |date=2021-06-12 }}. Accessed 2014-01-15.</ref>
|website                = {{URL|http://www.clustal.org/omega/}}
}}

'''Clustal''' is a [[computer program]] used for [[multiple sequence alignment]] in [[bioinformatics]].<ref name="Chenna2003">{{cite journal | vauthors = Chenna R, Sugawara H, Koike T, Lopez R, [[Toby Gibson|Gibson TJ]], [[Des Higgins|Higgins DG]], Thompson JD | title = Multiple sequence alignment with the Clustal series of programs | journal = Nucleic Acids Research | volume = 31 | issue = 13 | pages = 3497–500 | date = July 2003 | pmid = 12824352 | pmc = 168907 | doi = 10.1093/nar/gkg500 }}</ref> The software and its algorithms have gone through several iterations, with '''ClustalΩ (Omega)''' being the latest version {{As of|2011|lc=y}}. It is available as standalone software, via a [[Clustal#External links|web interface]], and through a server hosted by the [[European Bioinformatics Institute]].

Clustal has been an important bioinformatic software, with two of its academic publications amongst the top 100 papers cited of all time,  according to [[Nature (journal)|Nature]] in 2014.<ref>{{cite journal |vauthors=Van Noorden R, Maher B, [[Regina Nuzzo|Nuzzo R]] |date=October 2014 |title=The top 100 papers |journal=Nature |volume=514 |issue=7524 |pages=550–3 |bibcode=2014Natur.514..550V |doi=10.1038/514550a |pmid=25355343 |doi-access=free}}</ref> 
[[File:Multiple_Sequence_Alignment_Using_ClustalW.jpg|thumb|Multiple sequence alignment of [[Cyclin-dependent kinase 4|CDK4 protein]] generated with ClustalW. Arrows indicate [[Point mutation|point mutations]].]]

== History ==

=== Version history ===
* '''Clustal''': The original software for multiple sequence alignments, created by [[Desmond G. Higgins|Des Higgins]] in 1988, was based on deriving a guide tree from pairwise sequences of [[Amino acid|amino acids]] or [[Nucleotide|nucleotides]].<ref name="pmid3243435">{{cite journal | vauthors = Higgins DG, Sharp PM | title = CLUSTAL: a package for performing multiple sequence alignment on a microcomputer | journal = Gene | volume = 73 | issue = 1 | pages = 237–44 | date = December 1988 | pmid = 3243435 | doi = 10.1016/0378-1119(88)90330-7 }}</ref>
* '''ClustalV''': The second generation of Clustal, released in 1992. It introduced the ability to create new alignments from existing alignments in a process known as phylogenetic tree reconstruction. ClustalV also added the option to create [[Tree (data structure)|trees]] using the [[neighbor joining]] method.<ref name="pmid1591615">{{cite journal | vauthors = Higgins DG, Bleasby AJ, Fuchs R | title = CLUSTAL V: improved software for multiple sequence alignment | journal = Computer Applications in the Biosciences | volume = 8 | issue = 2 | pages = 189–91 | date = April 1992 | pmid = 1591615 | doi = 10.1093/bioinformatics/8.2.189 }}</ref>
*'''ClustalW''': The third generation, released in 1994. It improved upon the progressive alignment algorithm, including sequence [[weighting]] options based on [[Similarity (geometry)|similarity]] and [[Divergence (statistics)|divergence]]. Additionally, it added the option to run Clustal in [[Batch processing|batch mode]] from the [[Command-line interface|command line]].<ref>{{Cite journal |last1=Thompson |first1=J. D. |last2=Higgins |first2=D. G. |last3=Gibson |first3=T. J. |date=1994-11-11 |title=CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice |journal=Nucleic Acids Research |volume=22 |issue=22 |pages=4673–4680 |doi=10.1093/nar/22.22.4673 |issn=0305-1048 |pmid=7984417|pmc=308517 }}</ref>
*'''ClustalX''': Released in 1997, this was the first version to have a graphical user interface.<ref name="Thompson1997">{{cite journal | vauthors = Thompson JD, [[Toby Gibson|Gibson TJ]], Plewniak F, Jeanmougin F, Higgins DG | title = The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools | journal = Nucleic Acids Research | volume = 25 | issue = 24 | pages = 4876–82 | date = December 1997 | pmid = 9396791 | pmc = 147148 | doi = 10.1093/nar/25.24.4876 }}</ref>
*'''Clustal2''': This updated both ClustalW and ClustalX with higher accuracy and efficiency in 2007.<ref>{{Cite web|url=http://www.clustal.org/clustal2/|title=Clustal W and Clustal X Multiple Sequence Alignment|last=Dineen|first=David|website=www.clustal.org|access-date=2018-04-24|archive-date=2018-04-16|archive-url=https://web.archive.org/web/20180416130735/http://www.clustal.org/clustal2/|url-status=live}}</ref>
*'''ClustalΩ (Omega)''': The current version, released in 2011.<ref>{{Cite book|title=Multiple Sequence Alignment Methods|journal=<!--Bypass Citation bot -->|volume=1079|last1=Sievers|first1=Fabian|last2=Higgins|first2=Desmond G.|chapter=Clustal Omega, Accurate Alignment of Very Large Numbers of Sequences | name-list-style = vanc |date=2014-01-01|publisher=Humana Press|isbn=9781627036450|editor-last=Russell|editor-first=David J|series=Methods in Molecular Biology|pages=105–116|language=en|doi=10.1007/978-1-62703-646-7_6|pmid = 24170397}}</ref><ref>{{Cite book|volume=48|pages=3.13.1–16|last1=Sievers|first1=Fabian|last2=Higgins|first2=Desmond G.| name-list-style = vanc |date=2002-01-01|publisher=John Wiley & Sons, Inc.|isbn=9780471250951|language=en|doi=10.1002/0471250953.bi0313s48|pmid = 25501942|title=Clustal Omega|journal=Current Protocols in Bioinformatics|s2cid=1762688}}</ref>

=== Name origin ===
The guide tree in the initial versions of Clustal was constructed via a [[UPGMA]] cluster analysis of the pairwise alignments, hence the name CLUSTAL.<ref name="smbe">Des Higgins, presentation at the SMBE 2012 conference in Dublin.</ref><sup>cf.</sup><ref>{{cite journal | vauthors = Higgins DG, Sharp PM | title = CLUSTAL: a package for performing multiple sequence alignment on a microcomputer | journal = Gene | volume = 73 | issue = 1 | pages = 237–44 | date = December 1988 | pmid = 3243435 | doi = 10.1016/0378-1119(88)90330-7 | author-link2 = Paul M. Sharp }}</ref> The first four versions of Clustal were numbered using Arabic numerals (1 to 4), whereas the fifth version uses the Roman numeral V.<ref name="smbe" /><sup>cf.</sup><ref>{{cite journal | vauthors = Higgins DG, Sharp PM | title = Fast and sensitive multiple sequence alignments on a microcomputer | journal = Computer Applications in the Biosciences | volume = 5 | issue = 2 | pages = 151–3 | date = April 1989 | pmid = 2720464 | doi = 10.1093/bioinformatics/5.2.151 | author-link2 = Paul M. Sharp }}</ref><ref name="pmid1591615"/> The next two versions proceed alphabetically using the Latin alphabet, with W standing for weighted and X for [[X Window System|X Window]] to represent the changes introduced.<ref name="smbe" /><sup>cf.</sup><ref>{{cite journal | vauthors = Thompson JD, Higgins DG, [[Toby Gibson|Gibson TJ]] | title = CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice | journal = Nucleic Acids Research | volume = 22 | issue = 22 | pages = 4673–80 | date = November 1994 | pmid = 7984417 | pmc = 308517 | doi = 10.1093/nar/22.22.4673 }}</ref><ref name="Thompson1997"/> The name Omega was chosen to mark a change from the previous iterations.<ref name="smbe" />

== Function ==
Clustal aligns sequences using a [[heuristic]] that [[Iterative method|progressively]] builds a multiple sequence alignment from a set of pairwise alignments. This method works by analyzing the sequences as a whole and using the UPGMA/neighbor-joining method to generate a [[distance matrix]]. A guide tree is calculated from the scores of the sequences in the matrix, then subsequently used to build the multiple sequence alignment by progressively aligning the sequences in order of similarity.<ref name="CLUSTAL W Algorithm">{{Cite web | url=https://www-bimas.cit.nih.gov/clustalw/clustalw.html | title=CLUSTAL W Algorithm | access-date=2018-04-24 | archive-url=https://web.archive.org/web/20161201124147/https://www-bimas.cit.nih.gov/clustalw/clustalw.html | archive-date=2016-12-01 | url-status=dead }}</ref> 

Clustal creates multiple sequence alignments through three main steps:
#Complete a [[Sequence alignment|pairwise alignment]] using the progressive alignment method.
#Create a [[Molecular phylogenetics|guide tree]] (or use a user-defined tree).
#Use the guide tree to carry out a multiple alignment.

These steps are carried out automatically by the function "Do Complete Alignment". Other options are "Do Alignment from guide tree and phylogeny" and "Produce guide tree only".

=== Input/Output ===
This program accepts a wide range of input formats, including NBRF/[[Protein Information Resource|PIR]], [[FASTA format|FASTA]], EMBL/[[UniProt|Swiss-Prot]], Clustal, GCC/MSF, GCG9 RSF, and GDE.

The output format can be one or many of the following: Clustal, NBRF/[[Protein Information Resource|PIR]], [[GCG (software)|GCG]]/MSF, [[PHYLIP]], GDE, or NEXUS.
{| class="wikitable"
|+
Reading Multiple Sequence Alignment Output
!Symbol
!Definition
!Meaning
|-
|*
|asterisk
|positions that have a single and fully conserved residue
|-
|:
|colon
|conserved: conservation between groups of strongly similar properties (score&nbsp;&gt;&nbsp;0.5 on the [[Point accepted mutation#An example - PAM250|PAM 250]] matrix)
|-
|.
|period
|semi-conserved: conservation between groups of weakly similar properties (score&nbsp;&le;&nbsp;0.5 on the PAM 250 matrix)
|-
|
|blank
|non-conserved
|}
The same symbols are shown for both [[DNA]]/[[RNA]] alignments and [[protein]] alignments, so while * (asterisk) symbols are useful for both, the other consensus symbols should be ignored for DNA/RNA alignments.

=== Settings ===
The gap opening penalty and gap extension penalty parameters can be adjusted by the user.

== Clustal and ClustalV ==
=== Brief summary ===
The original Clustal software was developed in 1988 as a computational method for generating multiple sequence alignments on [[Personal computer|personal computers]]. ClustalV was released 4 years later and greatly improved upon the original software, adding and altering few key features. It was a full re-write, written in [[C (programming language)|C]] instead of [[Fortran]].

=== Algorithm ===
Both versions use the same fast [[Approximation algorithm|approximate algorithm]] to calculate the similarity scores between sequences, which in turn produces the pairwise alignments. The algorithm works by calculating the similarity scores as the number of [[Tuple|k-tuple]] matches between two sequences, accounting for a set penalty for gaps. The more similar the sequences, the higher the score. Once the sequences are scored, a [[dendrogram]] is generated through the UPGMA to generate an ordering of the multiple sequence alignment. Sequences are aligned in descending order by set order. This algorithm allows for very large data sets and is fast. However, the speed is dependent on the range of k-tuple matches selected for the particular sequence type.<ref name="aua.gr">{{Cite web |last=Higgins |first=Des |date=June 1991 |title=Clustal V Multiple Sequence Alignments. Documentation (Installation and Usage). |url=https://www.aua.gr/~eliop/mathimata/molevol/Askhsh1/clustalv.htm |url-status=live |access-date=2022-08-27 |website=www.aua.gr |archive-date=2023-04-12 |archive-url=https://web.archive.org/web/20230412000530/https://www.aua.gr/~eliop/mathimata/molevol/Askhsh1/clustalv.htm }}</ref>

=== Notable ClustalV improvements ===

Some of the most notable additions in ClustalV are profile alignments, and full command line interface options. The ability to use profile alignments allows the user to align two or more previous alignments or sequences to a new alignment and move misaligned sequences (low scored) further down the alignment order. This gives the user the option to gradually and methodically create multiple sequence alignments with more control than the basic option.<ref name="CLUSTAL W Algorithm"/> The option to run from the command line expedites the multiple sequence alignment process. Sequences can be run with a simple command,

<syntaxhighlight lang="c++"> clustalv nameoffile.seq </syntaxhighlight> or <syntaxhighlight lang="c++"> clustalv /infile=nameoffile.seq </syntaxhighlight>

and the program will determine what type of sequence it is analyzing. When the program is completed, the output of the multiple sequence alignment as well as the dendrogram go to files with .aln and .dnd extensions respectively. The command line interface uses the default parameters, and doesn't allow for other options.<ref name="aua.gr"/>

== ClustalW ==

=== Brief summary ===
[[File:Flowchart_for_ClustalW_Algorithm.svg|thumb|Depicts the steps the ClustalW software algorithm uses for global alignments]]
ClustalW, like other Clustal versions, is used for aligning multiple nucleotide or protein sequences efficiently. It uses progressive alignment methods, which prioritize sequences for alignment based on similarity until a global alignment is returned. ClustalW is a [[Matrix (mathematics)|matrix-based]] algorithm, whereas tools like [[T-Coffee]] and [[DIALIGN-T|Dialign]] are [[Consistency (statistics)|consistency-based]]. ClustalW is efficient, with competitive in comparison with similar software.{{Citation needed|date=April 2024}} This program requires three or more sequences in order to calculate a global alignment. For binary sequence alignment, other tools such as [[EMBOSS]] or [https://www.ebi.ac.uk/Tools/psa/lalign/ LALIGN] should be used.
[[File:Neighbor_Joining_Phylogenetic_Tree.png|thumb|Diagram showing neighbor-joining method in sequence alignment for bioinformatics]]

=== Algorithm ===
ClustalW uses progressive alignment algorithms. In these, sequences are aligned in most-to-least alignment score order. This heuristic is necessary to restrict the [[Time complexity|time-]] and [[Space complexity|memory-complexity]] required to find the [[Maximum and minimum|globally optimal solution]].

First, the algorithm computes a pairwise distance matrix between all pairs of sequences ([[Sequence alignment#Pairwise alignment|pairwise sequence alignment]]). Next, a [[Neighbor joining|neighbor-joining method]] uses midpoint rooting to create an overall guide tree.<ref>{{Cite web|url=https://www.megasoftware.net/web_help_7/hc_clustalw.htm|title=About CLUSTALW|website=www.megasoftware.net|access-date=2018-04-24|archive-date=2018-04-24|archive-url=https://web.archive.org/web/20180424202409/https://www.megasoftware.net/web_help_7/hc_clustalw.htm|url-status=live}}</ref> A diagram of this method is illustrated to the right. Finally, the guide tree is used as an approximate template to generate a global alignment.

=== Time complexity ===
ClustalW has a time complexity of <math>O(N^2)</math> because of its use of the neighbor-joining method. 

ClustalW2 added an option to use UPGMA instead which is faster for large input sizes. The command line flag in order to use it instead of neighbor-joining is:<syntaxhighlight lang="c++">
-clustering=UPGMA
</syntaxhighlight>As an approximate example, while a 10,000 sequences input would take over an hour for neighbor-joining, UPGMA would complete in less than a minute. 

ClustalW2 also added an iterative alignment accuracy. This option does not increase efficiency, but it does offer the ability to increase alignment accuracy. This can be especially useful for small datasets. 

The following flags activate iterative alignment: <syntaxhighlight lang="c++">
-Iteration=Alignment
-Iteration=Tree
-numiters

</syntaxhighlight>The first option refines the final alignment. The second option incorporates the scheme in the progressive alignment step. The third specifies the number of iteration cycles, where the default value is set to 3.<ref name=":2">{{Cite journal |last1=Larkin |first1=M.A. |last2=Blackshields |first2=G. |last3=Brown |first3=N.P. |last4=Chenna |first4=R. |last5=McGettigan |first5=P.A. |last6=McWilliam |first6=H. |last7=Valentin |first7=F. |last8=Wallace |first8=I.M. |last9=Wilm |first9=A. |date=2007-09-10 |title=Clustal W and Clustal X version 2.0 |journal=Bioinformatics |language=en |volume=23 |issue=21 |pages=2947–2948 |doi=10.1093/bioinformatics/btm404 |issn=1367-4803 |pmid=17846036 |doi-access=free}}</ref>

=== Accuracy and Results ===
The algorithm ClustalW uses is nearly optimal. It is most effective for datasets with a large degree of variance. On such datasets, the process of generating a guide tree is less sensitive to noise. ClustalW was one of the first multiple sequence alignment algorithms to combine pairwise alignment and global alignment to increase speed, but this decision reduces result accuracy. 

When multiple sequence alignment algorithms were compared in 2014, ClustalW was one of the fastest that was able to produce results at the desired level of accuracy. However, it was not as accurate as consistency-based competitors such as T-Coffee.<ref name=":3">{{cite journal | vauthors = Pais FS, Ruy PC, Oliveira G, Coimbra RS | title = Assessing the efficiency of multiple sequence alignment programs | journal = Algorithms for Molecular Biology | volume = 9 | issue = 1 | pages = 4 | date = March 2014 | pmid = 24602402 | pmc = 4015676 | doi = 10.1186/1748-7188-9-4 | doi-access = free }}</ref> Out of MAFFT, T-Coffee, and Clustal Omega, ClustalW has the lowest accuracy for full-length sequences, but its accuracy is still considered acceptable. Additionally, ClustalW was the most memory-efficient algorithm of those studied.<ref name=":3" /> Continued updates to the software have made ClustalW2 more accurate while maintaining this speed.<ref name=":2" />

== Clustal Omega ==

=== Brief summary ===
[[File:Clustal_Omega_Algorithm_Flowchart.svg|thumb|Flowchart depicting the step-by-step algorithm used in Clustal Omega.]]
'''ClustalΩ''' (alternatively written as '''Clustal O''' and '''Clustal Omega''') is a fast and [[Scalability|scalable]] program written in C and [[C++]] used for multiple sequence alignment. It uses seeded guide trees and a new [[Hidden Markov model|HMM]] engine that focuses on two profiles to generate these alignments.<ref>{{Cite web|url=https://www.ebi.ac.uk/Tools/msa/clustalo/|title=Clustal Omega < Multiple Sequence Alignment < EMBL-EBI|last=EMBL-EBI|website=www.ebi.ac.uk|language=en|access-date=2018-04-18|archive-date=2018-04-29|archive-url=https://web.archive.org/web/20180429093325/https://www.ebi.ac.uk/Tools/msa/clustalo/|url-status=live}}</ref><ref>{{Cite web|url=http://www.clustal.org/|title=Clustal Omega, ClustalW and ClustalX Multiple Sequence Alignment|last=Dineen|first=David|website=www.clustal.org|access-date=2018-04-18|archive-date=2010-05-29|archive-url=https://web.archive.org/web/20100529100824/http://www.clustal.org/|url-status=live}}</ref> The program requires three or more sequences in order to calculate the [[multiple sequence alignment]]. Clustal Omega is consistency-based and is widely viewed{{By whom|date=April 2024}} as one of the fastest online implementations of all multiple sequence alignment tools and still ranks high in accuracy, among both consistency-based and matrix-based algorithms.

=== Algorithm ===
[[File:Profile_Hidden_Markov_Model.png|thumb|The structure of a profile HMM used in the implementation of Clustal Omega is shown here.]]
Clustal Omega has five main steps in order to generate the [[multiple sequence alignment]]. 

# A pairwise alignment is produced using the k-tuple method.This is a heuristic method that isn't guaranteed to find an optimal solution, but is more efficient than using [[dynamic programming]].
# Sequences are clustered using the modified mBed method.<ref name=":0" /> The mBed method calculates pairwise distance using sequence embedding.
# The [[K-means clustering|k-means clustering method]] is applied.
# A guide tree is constructed using the [[UPGMA|UPGMA method]]. In the figure to the right, this is shown as multiple guide tree steps leading into one final guide tree construction because of the agglomerative nature of UPGMA. At each step (diamonds in the flowchart), the nearest two clusters are combined. This is repeated until a final, global tree can be assessed. 
# The final [[multiple sequence alignment]] is produced with the HHAlign package from the [[HH-suite|HH-Suite]] using two profile [[Hidden Markov model|HMM's]]. A profile HMM is a linear state machine consisting of a series of nodes, each of which corresponds roughly to a position (column) in the alignment from which it was built.<ref>{{Cite web |title=Profile HMM Analysis |url=http://www.biology.wustl.edu/gcg/hmmanalysis.html |url-status=dead |archive-url=https://web.archive.org/web/20190724174527/http://www.biology.wustl.edu/gcg/hmmanalysis.html |archive-date=2019-07-24 |access-date=2018-05-01 |website=www.biology.wustl.edu}}</ref>

=== Time complexity ===
The time complexity of exactly computing an optimal alignment of <math>N</math>sequences of length <math>L</math> is  <math>O(L^N)</math> which is prohibitive for even a small number of sequences. To manage this, Clustal Omega uses a modified version of mBed which has a complexity of <math>O(N \log N) </math>,<ref name=":0">{{cite journal | vauthors = Blackshields G, Sievers F, Shi W, Wilm A, Higgins DG | title = Sequence embedding for fast construction of guide trees for multiple sequence alignment | journal = Algorithms for Molecular Biology | volume = 5 | pages = 21 | date = May 2010 | pmid = 20470396 | pmc = 2893182 | doi = 10.1186/1748-7188-5-21 | doi-access = free }}</ref><ref>{{cite journal | vauthors = Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, Thompson JD, Higgins DG | title = Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega | journal = Molecular Systems Biology | volume = 7 | issue = 1 | pages = 539 | date = October 2011 | pmid = 21988835 | pmc = 3261699 | doi = 10.1038/msb.2011.75 }}</ref> and produces guide trees that are as accurate as those from conventional methods. The speed and accuracy of the guide trees in Clustal Omega is attributed to the implementation of a modified mBed algorithm. It also reduces the computational time and memory requirements to complete alignments on large datasets.

=== Accuracy and results ===
The accuracy of Clustal Omega on a small number of sequences is, on average, very similar to what are considered high quality sequence aligners.{{Examples needed|date=April 2024}} On extremely large datasets with hundreds of thousands of input sequences, Clustal Omega outperforms all other algorithms in time, memory, and accuracy of results.<ref name=":1">{{Cite journal|last1=Daugelaite|first1=Jurate|last2=O' Driscoll|first2=Aisling|last3=Sleator|first3=Roy D. | name-list-style = vanc |date=2013|title=An Overview of Multiple Sequence Alignments and Cloud Computing in Bioinformatics|journal=ISRN Biomathematics|language=en|volume=2013|pages=1–14|doi=10.1155/2013/615630|issn=2090-7702|doi-access=free}}</ref> It is capable of running 100,000+ sequences on one processor in a few hours.

Clustal Omega uses the HHAlign package of the HH-Suite, which aligns two profile Hidden Markov Models instead of a profile-profile comparison. This improves the quality of the sensitivity and alignment significantly.<ref name=":1" /> This, combined with the mBed method, gives Clustal Omega its advantage over other sequence aligners.

On data sets with non-conserved terminal bases, Clustal Omega can be more accurate than [[ProbCons|Probcons]] or [[T-Coffee]], despite the fact that both are consistency-based algorithms. On an efficiency test with programs that produce high accuracy scores, [[MAFFT]] was the fastest, closely followed by Clustal Omega. Both were faster than T-Coffee, however [[MAFFT]] and Clustal Omega required more memory to run.<ref name=":3" />

== Clustal2 (ClustalW/ClustalX) ==

[http://www.clustal.org/clustal2/ Clustal2] is the packaged release of both the command-line ClustalW and graphical Clustal X. Neither are new tools, but are updated and improved versions of the previous implementations seen above. Both downloads come pre-compiled for many operating systems like Linux, Mac OS X and Windows (both XP and Vista). This release was designed to make the website more organized and user friendly, as well as updating the source codes to their most recent versions. Clustal2 is version 2 of both ClustalW and ClustalX, which is where it gets its name. Past versions can still be found on the website, however, every pre-compilation is now up to date.

== See also ==
{{Div col|colwidth=22em}}
* [[Sequence alignment software]]
* [[Sequence mining]]
* [[T-Coffee]]
* [[Align-m]]
* [[DIALIGN-T]]
* [[DIALIGN-TX]]
* [[JAligner]]
* [[MAFFT]]
* [[MAVID]]
* [[MUSCLE (alignment software)|MUSCLE]]
* [[ProbCons]]
{{div col end}}

== References ==
{{Reflist|2}}

== External links ==
*[http://www.clustal.org Clustal Homepage]  (free Unix/Linux, Mac, and Windows download)
*[http://www.ebi.ac.uk/Tools/msa/clustalo/ Clustal Omega mirror at the EBI]

{{Bioinformatics}}

[[Category:Phylogenetics software]]