the computation of an accurate multiple sequence alignment ( msa ) is central to a large number of bioinformatics analyses , ranging from phylogeny , profile construction , structure prediction and more recently sequence / structure activity relationship . despite its importance
, the msa problem has not yet met with a definitive answer and a wide variety of alternative methods are currently available ( 3,4 ) .
all these methods are meant to address the same problem in different ways . in recent years
, many efforts have been undertaken to characterize their relative accuracy but the overall outcome suggests that there is no such thing as a perfect msa method , with each individual method having specific strengths and weaknesses . in practice , evaluation is made using structure - based msas as a standard of truth and the expected accuracy of a method is deduced from its ability to produce a structurally correct sequence alignment while using sequence information only .
at least five such collections of reference alignments ( 58 ) have been established , and although some methods give better average results than others , one can not know in advance which method will outperform the others on a given dataset . as such , it is always possible for the worst method to outperform all the others on a specific dataset . for the biologist , this makes it impossible to use anything other than a weak statistical argument ( i.e. best method on average ) to choose one method among the others when computing an alignment .
the design of meta - methods ( or jury - based methods ) is one way of addressing such situations in biology .
meta - methods are meant to combine the output of several alternative methods into one final output .
they are based on the empirical reasoning that errors produced by independent prediction systems should not be consistent , therefore suggesting agreement as an indication of correctness .
such an approach was successfully used in the field of gene predictions ( 9 ) or for secondary structure predictions ( 10 ) . combining alignments , however , is less simple than building consensus prediction and it is only in 1999 that an effective strategy was proposed by bucka - lassen ( 11 ) .
an alternative to the bucka - lassen strategy , using consistency , was later introduced in the t - coffee ( 1 ) algorithm .
recently , this algorithm was further modified in order to address the problem of combining alternative msas into one ( 2 ) .
t - coffee ( 1 ) is a progressive consistency - based algorithm that compiles an alignment on the basis of its consistency with a collection of pairwise constraints . in practice ,
the constraints correspond to pairs of residues that could end up aligned in the final alignment .
these constraints , however , are not necessarily all compatible with one another and the goal of the algorithm is to fit as many as possible within the final alignment , while discarding those that were hopefully biologically less relevant .
the term consistency refers to the notion that one tries to compute the alignment having the highest possible consistency with the constraint list .
this notion was introduced by gotoh ( 12 ) and later re - used in several algorithms ( 13,14 ) . in 2000 , notredame et al .
( 1 ) described a variation of the progressive algorithm using consistency as a scoring scheme .
this combination proved quite successful and is now at the core of several msa packages ( 1517 ) . in its default mode , t - coffee uses , as a list of constraints , all the pair - wise matches extracted from a compilation of all possible global pair - wise alignments and the 10 best local alignments from each pair of sequences . yet , this is merely one of the possible recipes to assemble such a list of constraints , and alternatives are possible .
for instance , probcons ( 16 ) uses suboptimal pairwise global alignments ( as emitted by an hmm with posterior decoding ) ; pcma ( 15 ) uses pairwise profile comparisons and expresso ( 18 ) uses a mixture of sequence and structure - based alignments .
following the same principle , it is also possible to generate alternative msas and compile them into a single list of constraints .
this latest approach forms the basis of m - coffee ( 2 ) , where eight msa methods are used to generate alternative msas .
extensive benchmarking showed that this combination results in a modest but consistent improvement over each individual method , with m - coffee producing the best scoring alignment on two of three of the datasets contained in balibase ( 5 ) , prefab ( 6 ) and homstrad ( 2 ) .
another interesting by - product of alignment combination is the possibility of estimating the local consistency between the final alignment and the individual alignments .
this amounts to measuring , for every residue , the fraction of individual alignments that support its position in the final alignment .
this measure is named the core index ( consistency of overall residue evaluation ) and was shown to be very informative with respect to the overall alignment accuracy ( 19 ) .
these initial reports recently gained further support thanks to some extensive analysis carried out by sonhammer et al.(20 ) whose results indicate that the consistency between an msa and a pre - computed collection of alternative alignments gives very reliable information with respect to the structural correctness of that alignment . as such , the local consistency measure appears to be one of the most reliable predictors of alignment accuracy available today .
the server we present here computes an alignment with eight of the most commonly used msa packages .
it then outputs a consensus alignment along with a core - based local evaluation that can either be color - coded or ascii based .
two mirrors of these services currently run on separate clusters : one at the swiss institute of bioinformatics on the vital - it framework , the other at the cnrs in marseilles , france .
both mirrors can be accessed via the t - coffee homepage : www.tcoffee.org and extra mirrors should be added in the close future .
the principle of m - coffee is to compute several alternative multiple alignments in order to combine them into one consensus alignment . by default ,
eight methods were chosen for this purpose : pcma ( version 2.0 ) ( 15 ) , poa ( version 2.0 ) ( 21 ) , dialign - t ( version 0.2.1 ) ( 22 ) , mafft ( version 5.431 , l - ins - i ) ( 17 ) , muscle ( version 3.6 ) , probcons ( version 1.2 ) , clustalw ( 23 ) and t - coffee ( 1 ) . apart from mafft that is used in its most accurate mode ( mafft-  - localpair-  -maxiterate 1000 )
this produces an msa that is then turned into a t - coffee primary library .
all these libraries are then combined in order to generate an msa . in order to compute the final alignment , the server runs the following command : t_coffee < seq > -method poa_msa , dialignt_msa , mafft_msa , clustalw_msa , muscle_msa , probcons_msa , t_coffee_msa , pcma_msa .
the regular mode merely requires the user to cut and paste a set of sequences in fasta format . the advanced mode ( figure 1 ) offers more possibilities and guides the user with a series of bulleted points : 
 cut and paste your sequences .
for instance , selecting only lalign_id_pair and slow_pair will lead to the computation of a regular t - coffee msa .
selecting several methods ( or all of them , as in the regular mode displayed on figure 1 ) will lead to a consensus t - coffee msa .
if the msa method one wants to combine is missing on this form , another server named 
combine server works on the same principle as m - coffee but does not compute the msas itself and requires the user to cut and paste pre - computed msas . at this point
it should be used if one wants to incorporate specific constraints or structure - based sequence alignments.output .
the most notable element is score_html that will cause the server to produce a colored version of the final alignment ( figure 2 ) . in this output , residues are individually colored according to the consistency of their alignment with the t - coffee library .
residues in red are in perfect agreement with every constituting multiple alignment while those in blue have the lowest agreement ( i.e. the lowest support in the individual msas ) .
previous analysis indicates that 90% of the residues having a score of 7 or higher ( dark yellow , orange and red ) are correctly aligned ( 24 ) .
a text version of this output is available as score_ascii where each residue is replaced with its consistency estimation on a scale between 0 and 9 ( 9 corresponding to the red - brick residues in the color - output ) .
these score_ascii files can be used to process multiple alignments ( block extraction ) using seq_reformat , one of the utilities distributed along with t - coffee . for this purpose
, users can download their alignment , the score_ascii file and use the command line version of t - coffee with the following syntax : 
 figure 1.method selection on the advanced m - coffee server form .
each check box corresponds to either a pairwise ( _ pair ) or a multiple sequence alignment method ( _ msa ) .
correctly aligned residues ( as judged from the reference ) are in upper case , non - correct ones are in lower case . in this colored output ,
each residue has a color that indicates the agreement of the individual msas with respect to the alignment of that specific residue .
dark red indicates residues aligned in a similar fashion among all the individual msas ; blue indicates a very low agreement .
for instance , selecting only lalign_id_pair and slow_pair will lead to the computation of a regular t - coffee msa .
selecting several methods ( or all of them , as in the regular mode displayed on figure 1 ) will lead to a consensus t - coffee msa . if the msa method one wants to combine is missing on this form , another server named 
combine server works on the same principle as m - coffee but does not compute the msas itself and requires the user to cut and paste pre - computed msas . at this point
it should be used if one wants to incorporate specific constraints or structure - based sequence alignments .
the most notable element is score_html that will cause the server to produce a colored version of the final alignment ( figure 2 ) . in this output ,
residues are individually colored according to the consistency of their alignment with the t - coffee library .
residues in red are in perfect agreement with every constituting multiple alignment while those in blue have the lowest agreement ( i.e. the lowest support in the individual msas ) .
previous analysis indicates that 90% of the residues having a score of 7 or higher ( dark yellow , orange and red ) are correctly aligned ( 24 ) .
a text version of this output is available as score_ascii where each residue is replaced with its consistency estimation on a scale between 0 and 9 ( 9 corresponding to the red - brick residues in the color - output ) .
these score_ascii files can be used to process multiple alignments ( block extraction ) using seq_reformat , one of the utilities distributed along with t - coffee . for this purpose
, users can download their alignment , the score_ascii file and use the command line version of t - coffee with the following syntax : method selection on the advanced m - coffee server form .
each check box corresponds to either a pairwise ( _ pair ) or a multiple sequence alignment method ( _ msa ) .
correctly aligned residues ( as judged from the reference ) are in upper case , non - correct ones are in lower case . in this colored output
, each residue has a color that indicates the agreement of the individual msas with respect to the alignment of that specific residue .
dark red indicates residues aligned in a similar fashion among all the individual msas ; blue indicates a very low agreement .
dark yellow , orange and red residues can be considered to be reliably aligned . 
-struc_in_f number_aln -action + keep  [ 5 - 9] where < aln > is the name of the alignment and < score_ascii > the name of the score_asccii file .
this syntax will replace by a gap (  - ' ) every residue having an ascii_score lower than 5 ( green and blue residues on the colored output ) .
the principle of m - coffee is to compute several alternative multiple alignments in order to combine them into one consensus alignment . by default ,
eight methods were chosen for this purpose : pcma ( version 2.0 ) ( 15 ) , poa ( version 2.0 ) ( 21 ) , dialign - t ( version 0.2.1 ) ( 22 ) , mafft ( version 5.431 , l - ins - i ) ( 17 ) , muscle ( version 3.6 ) , probcons ( version 1.2 ) , clustalw ( 23 ) and t - coffee ( 1 ) . apart from mafft that is used in its most accurate mode ( mafft-  - localpair-  -maxiterate 1000 )
this produces an msa that is then turned into a t - coffee primary library .
in order to compute the final alignment , the server runs the following command : t_coffee < seq > -method poa_msa , dialignt_msa , mafft_msa , clustalw_msa , muscle_msa , probcons_msa , t_coffee_msa , pcma_msa .
the regular mode merely requires the user to cut and paste a set of sequences in fasta format . the advanced mode ( figure 1 ) offers more possibilities and guides the user with a series of bulleted points : 
 cut and paste your sequences .
for instance , selecting only lalign_id_pair and slow_pair will lead to the computation of a regular t - coffee msa .
selecting several methods ( or all of them , as in the regular mode displayed on figure 1 ) will lead to a consensus t - coffee msa . if the msa method one wants to combine is missing on this form , another server named 
combine server works on the same principle as m - coffee but does not compute the msas itself and requires the user to cut and paste pre - computed msas . at this point
it should be used if one wants to incorporate specific constraints or structure - based sequence alignments.output .
the most notable element is score_html that will cause the server to produce a colored version of the final alignment ( figure 2 ) . in this output , residues are individually colored according to the consistency of their alignment with the t - coffee library .
residues in red are in perfect agreement with every constituting multiple alignment while those in blue have the lowest agreement ( i.e. the lowest support in the individual msas ) .
previous analysis indicates that 90% of the residues having a score of 7 or higher ( dark yellow , orange and red ) are correctly aligned ( 24 ) .
a text version of this output is available as score_ascii where each residue is replaced with its consistency estimation on a scale between 0 and 9 ( 9 corresponding to the red - brick residues in the color - output ) .
these score_ascii files can be used to process multiple alignments ( block extraction ) using seq_reformat , one of the utilities distributed along with t - coffee . for this purpose , users can download their alignment , the score_ascii file and use the command line version of t - coffee with the following syntax : 
 figure 1.method selection on the advanced m - coffee server form .
each check box corresponds to either a pairwise ( _ pair ) or a multiple sequence alignment method ( _ msa ) .
correctly aligned residues ( as judged from the reference ) are in upper case , non - correct ones are in lower case . in this colored output ,
each residue has a color that indicates the agreement of the individual msas with respect to the alignment of that specific residue .
dark red indicates residues aligned in a similar fashion among all the individual msas ; blue indicates a very low agreement .
duplicated names are now supported although not recommended . alignment computation . this section defines the way the primary library is computed .
for instance , selecting only lalign_id_pair and slow_pair will lead to the computation of a regular t - coffee msa .
selecting several methods ( or all of them , as in the regular mode displayed on figure 1 ) will lead to a consensus t - coffee msa . if the msa method one wants to combine is missing on this form , another server named 
combine server works on the same principle as m - coffee but does not compute the msas itself and requires the user to cut and paste pre - computed msas . at this point
it should be used if one wants to incorporate specific constraints or structure - based sequence alignments .
the most notable element is score_html that will cause the server to produce a colored version of the final alignment ( figure 2 ) . in this output
, residues are individually colored according to the consistency of their alignment with the t - coffee library .
residues in red are in perfect agreement with every constituting multiple alignment while those in blue have the lowest agreement ( i.e. the lowest support in the individual msas ) .
previous analysis indicates that 90% of the residues having a score of 7 or higher ( dark yellow , orange and red ) are correctly aligned ( 24 ) .
a text version of this output is available as score_ascii where each residue is replaced with its consistency estimation on a scale between 0 and 9 ( 9 corresponding to the red - brick residues in the color - output ) .
these score_ascii files can be used to process multiple alignments ( block extraction ) using seq_reformat , one of the utilities distributed along with t - coffee . for this purpose , users can download their alignment , the score_ascii file and use the command line version of t - coffee with the following syntax : method selection on the advanced m - coffee server form .
each check box corresponds to either a pairwise ( _ pair ) or a multiple sequence alignment method ( _ msa ) .
correctly aligned residues ( as judged from the reference ) are in upper case , non - correct ones are in lower case . in this colored output , each residue has a color that indicates the agreement of the individual msas with respect to the alignment of that specific residue .
dark red indicates residues aligned in a similar fashion among all the individual msas ; blue indicates a very low agreement .
dark yellow , orange and red residues can be considered to be reliably aligned . 
-struc_in_f number_aln -action + keep  [ 5 - 9] where < aln > is the name of the alignment and < score_ascii > the name of the score_asccii file .
this syntax will replace by a gap (  - ' ) every residue having an ascii_score lower than 5 ( green and blue residues on the colored output ) .
m - coffee provides biologists with a useful alternative to the a priori choice of an msa method . although m - coffee does not entirely solve the question of which method should be used , its local scoring scheme makes it easier to read the alignment and determine which portions are the most informative .
further developments will include making more methods available , as well as making it possible to combine sequences and structures , using the expresso protocol .
 the m - coffee server is a web server that makes it possible to compute multiple sequence alignments ( msas ) by running several msa methods and combining their output into one single model . 
 this allows the user to simultaneously run all his methods of choice without having to arbitrarily choose one of them . 
 the msa is delivered along with a local estimation of its consistency with the individual msas it was derived from . 
 the computation of the consensus multiple alignment is carried out using a special mode of the t - coffee package [ notredame , higgins and heringa ( t - coffee : a novel method for fast and accurate multiple sequence alignment . 
 j. mol . 
 biol . 
 2000 ; 302 : 205217 ) ; wallace , o'sullivan , higgins and notredame ( m - coffee : combining multiple sequence alignment methods with t - coffee . 
 nucleic acids res . 
 2006 ; 34 : 16921699 ) ] given a set of sequences ( dna or proteins ) in fasta format , m - coffee delivers a multiple alignment in the most common formats . 
 m - coffee is a freeware open source package distributed under a gpl license and it is available either as a standalone package or as a web service from www.tcoffee.org . 
INTRODUCTION
METHODS
Primary library: computation of the initial MSAs
M-Coffee alignment computation
Using the M-Coffee server
CONCLUSION AND FUTURE DEVELOPMENTS