The server can be accessed at www.tcoffee.org. Following the M-Coffee link will either take the user to the regular or advanced mode. 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. Sequences should be in FASTA format. 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. The lower section (xxx_msa) displays the list of available MSA methods. Selecting only one of these methods will generate the corresponding alignment. 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’ should be used (accessible from www.tcoffee.org). The ‘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 Output section makes it possible to control the output format. 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 (link)). 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). Users should choose their methods of choice in order to combine them.

Typical colored output. This output was obtained by using the kinase1_ref5 from BaliBase. 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.

        t_coffee -other_pg seq_reformat -in -struc_in -struc_in_f number_aln -action +keep ‘[5-9]’
Where is the name of the alignment and 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).