(A) Example of an AMSA. Protein sequences containing symbols from the amino acid alphabet and ‘-’ to represent gaps are represented in the standard MSA format at the beginning of the file. Annotation layers (layers beginning with ‘#’) are in the second part of the AMSA file and they contain any ad hoc alphabet. The meaning of each symbol of the alphabet is described by the symbol ∼ value pairs in the description field of each layer. Note that the symbol ‘-’ in annotation denotes a default value described by the DEFAULT key. Annotation attached to the individual sequences is represented as key = value pairs (e.g. weight of the sequence). Annotation attached to individual residues of a sequence is described in a cross-referenced layer: in the example the ‘ss_layer’ cross-reference of THM1_THADA points to the secondary structure of the protein in layer #_SS_THM1_THADA. MSA column annotation is used to label disulfide bridges (#_SITE_DISULFIDE) where numbers represent the cysteine couple and values represent the expected symbol (C for cysteine in this example). The remaining layers represent parameters of the profile: the topology of the model (#_LABEL_), the two matrices used to generate pseudocounts (layers #_MATRIX_ and #_MATRIX_2) with the respective position-specific weights of the pseudocounts. (B) The same alignment viewed in Jalview.

(A) AMSA 1.0 grammar in Extended Backus-Naur Form. Explanation of the grammar symbols: ‘=’ assign operator; ‘()’ grouping operator; ‘[]’ optional (0 or 1 occurrence); ‘{}’ repetition (0 or more occurrences); ‘-’ exclude following symbol; ‘|’ alternation (or), ‘??’ special sequence. Note that any visible symbol is accepted for sequence and annotation alphabets, except the special symbols ‘#’, ‘=’, ‘∼’ and ‘>’. (B) Recommendations for the AMSA 1.0 format. An AMSA file consists of aligned sequences and annotation represented in FASTA format. Is possible to precede the sequence and annotation block with a version header followed by general comments (lines starting with '#') and global annotation (lines starting with ‘##’ followed by a key = text); in this case, we must terminate the sequence and annotation block using symbols ‘#//’. Any ad hoc alphabet can be used to describe sequences and annotation. The meaning of the alphabet used by an annotation layer can be explicitly represented as pairs value ∼ symbol in its description field. Sequence- (or annotation)-specific annotation is added after the ID field using key = value (or key = ‘text with white-spaces’). Sequence residues are annotated using a cross-reference to an annotation layer (key = # residue_annotation_ID). Though not required by the specification, we suggest to include the reference sequence ID in the residue_annotation_ID to facilitate the visual mapping from annotation to sequence. Columns of the MSA are annotated using annotation layers not linked to one or multiple sequences (see Figure 2 for an example). Two keywords are reserved in the current format: ‘DEFAULT’ is used to set a default value for the ‘-’ symbol of the annotation sequence; ‘SYM_LEN’ defines the number of characters required to encode a symbol. The SYM_LEN = digits permits to extend the format of sequences and annotations to any alphabet, e.g. codons (SYM_LEN = 3), to store PDB coordinates (SYM_LEN = 8 to store up to 6 digits, dot and sign), etc. If SYM_LEN is not specified, its default value should be 1 (one character per symbol). Note that we can achieve the same result by encoding multi-character symbols on a corresponding number of layers, this having the advantage that standard MSA editors can accept the format.