An alignment format defines the permitted layout and content of text in a file. This includes required text tokens and formatting conventions. Typically, the aligned sequences, sequence identifier codes and sequence position numbers are given in some form. Non-printable control characters do not occur in any of the common alignment formats, making most of the formats suitable for viewing on screen or for printing out.

"clustal" is probably the most commonly used alignment format. It is generated by the Clustalw multiple sequence alignment program. Many variants of this format are in common use. The first line must begin with the text CLUSTAL. The alignment is given in blocks of 50 residues with the aligned sequences appearing under each other. There are no sequence position numbers. Each sequence line begins with the sequence identifier code. For example:

CLUSTAL W(1.83) multiple sequence alignment


IXI_234         TSPASIRPPAGPSSRPAMVSSRRTRPSPPGPRRPTGRPCCSAAPRRPQAT
IXI_235         TSPASIRPPAGPSSR---------RPSPPGPRRPTGRPCCSAAPRRPQAT
IXI_236         TSPASIRPPAGPSSRPAMVSSR--RPSPPPPRRPPGRPCCSAAPPRPQAT
IXI_237         TSPASLRPPAGPSSRPAMVSSRR-RPSPPGPRRPT----CSAAPRRPQAT
                                                                  

IXI_234         GGWKTCSGTCTTSTSTRHRGRSGWSARTTTAACLRASRKSMRAACSRSAG
IXI_235         GGWKTCSGTCTTSTSTRHRGRSGW----------RASRKSMRAACSRSAG
IXI_236         GGWKTCSGTCTTSTSTRHRGRSGWSARTTTAACLRASRKSMRAACSR--G
IXI_237         GGYKTCSGTCTTSTSTRHRGRSGYSARTTTAACLRASRKSMRAACSR--G
                                                                  

IXI_234         SRPNRFAPTLMSSCITSTTGPPAWAGDRSHE
IXI_235         SRPNRFAPTLMSSCITSTTGPPAWAGDRSHE
IXI_236         SRPPRFAPPLMSSCITSTTGPPPPAGDRSHE
IXI_237         SRPNRFAPTLMSSCLTSTTGPPAYAGDRSHE

Beyond "clustal", the most widespread alignment formats are those generated by other popular programs:

In all the alignment formats except MSF, gaps inserted into the sequence during the alignment are indicated by the '-' character. In contrast, MSF format uses '.' (full stop) for gaps inside a sequence and '~' (tilde) for gaps at the ends of an alignment.

When editing alignments it is possible to use any text editor that is capable of writing files in plain text format. The drawback is that it is very easy to unwittingly break some rules of the format. Therefore, if you intend to manipulate or edit alignments substantially, investigate using a full-blown alignment editor such as mse. Such editors should have an option to save the alignment to a file in one or more of the standard formats.

Some alignment formats can hold only a pair of sequences (pairwise alignment) whereas others can hold multiple sequences (multiple sequence alignment). You should never use a pairwise alignment format to hold a multiple sequence alignment as the file would be unparsable by EMBOSS and other systems.

An alignment can be read from a file in any of the standard sequence formats that are suitable for alignments. For descriptions and examples of the supported formats see Section A.3, “Supported Alignment Formats”.

The supported alignment formats are summarised in the table below. The columns are as follows: Output format (format name), Nuc ("true" indicates nucleotide sequence data may be represented), Pro ("true" indicates protein sequence data may be represented, Header (whether the standard EMBOSS alignment header is included), Minseq (minimum sequences in alignment), Maxseq (maximum sequences in alignment) and Description (short description of the format).

All alignment formats excluding those (FASTA, MSF) that are also standard sequence formats, have a block of information (comments) at the start of the alignment describing the program, date, output filename, ID names of the sequences and some of the parameters and statistics of the alignment. For example:

########################################
# Program:  demoalign
# Rundate:  Thu Jan 17 09:30:08 2002
# Report_file: stdout
########################################
#=======================================
#
# Aligned_sequences: 4
# 1: IXI_234
# 2: IXI_235
# 3: IXI_236
# 4: IXI_237
# Matrix: EBLOSUM62
# Gap_penalty: 9
# Extend_penalty: -1
#
# Length: 131
# Identity:      95/131 (72.5%)
# Similarity:   127/131 (96.9%)
# Gaps:          25/131 (19.1%)
# Score:  100.0
#
#=======================================   

Length. 

# Length: 131

This line gives the length of the alignment, including any gaps that have been introduced to construct the alignment.

Identity. 

# Identity:      95/131 (72.5%)

This line gives the number of positions (95) over the length of the alignment where all of the residues or bases at that position are identical. The alignment length (131) and the percentage of positions (72.5%) in the alignment where there are such identities are shown.

Similarity. 

# Similarity:   127/131 (96.9%)

This line gives the number of positions over the length of the alignment where over 50% of the residues or bases at that position are similar to one another. Two residues or bases are defined as similar when their comparison score, as defined by the comparison matrix used by the alignment program, has a positive score. Again the alignment length (131) and the percentage (96.9%) of positions in the alignment where there are similarities is indicated.

Gaps. 

# Gaps:          25/131 (19.1%)

This line gives the number of positions over the length of the alignment containing one or more gap characters. The alignment length (131) and percentage (19.1%) of positions in the alignment where there are gaps is given.

Score. 

# Score: 100.0

This line gives the score for the alignment. The value depends on the alignment program, particularly the comparison matrix and gap penalties, and of course the sequences that were aligned. For an explanation of the score schemes refer to the relevant application documentation.

Alignment inputs are referred to on the EMBOSS command line by their USA (Section 6.6, “The Uniform Sequence Address (USA)”). This is a standard sequence naming scheme used by all EMBOSS applications. In the case of alignments, a USA specifies two or more sequences that should be read from a file. Other sequence sources such as an application or a web server can also be specified.

Alignment outputs, in contrast, are not specified by a USA. If you want to write an alignment to a file in one of the standard alignment formats, you must specify a simple name for the file as you would for a standard output file.

You may also write aligned sequences to a file in one of the standard sequences formats (Section A.1, “Supported Sequence Formats”). These are the formats to choose when the sequences are to be used by another EMBOSS application. In such cases, the sequences given will still be in their aligned condition (i.e. may include gap characters) but the alignment will not be obvious as the sequences will not be lined up and sequence positions may not be indicated.

There are also a set of command line qualifiers (Section 6.4, “Datatype-specific Command Line Qualifiers”) that are used for alignment input and output. These allow you to set such things as the file name and format and the records to print to the standard alignment header (see above).

For example, here the water application is called on two input sequences (Seq1.seq and Seq2.seq) to generate an alignment output file (SeqOut.seq) in MSF format:

Various programs for handling alignments (xref linkend="FormatsAlignmentIntroApps" />) are provided and are organised into groups of related functionality (xref linkend="FormatsAlignmentIntroGroups" />). These are either part of the main package or are included under the EMBASSY grouping (xref linkend="FormatsAlignmentIntroPackages" />).

Application Groups for Alignments

Alignment Applications

EMBASSY Packages for Alignments