Multiple Sequence Alignments (MSA)

ClustalW format

The ClustalW format is a relatively simple text file containing a single multiple sequence alignment of DNA, RNA, or protein sequences. It was first used as an output format for the clustalw programs, but nowadays it may also be generated by various other sequence alignment tools. The specification is straight forward:

  • The first line starts with the words:



  • After the above header there is at least one empty line

  • Finally, one or more blocks of sequence data are following, where each block is separated by at least one empty line.

Each line in a blocks of sequence data consists of the sequence name followed by the sequence symbols, separated by at least one whitespace character. Usually, the length of a sequence in one block does not exceed 60 symbols. Optionally, an additional whitespace separated cumulative residue count may follow the sequence symbols. Optionally, a block may be followed by a line depicting the degree of conservation of the respective alignment columns.


Sequence names and the sequences must not contain whitespace characters! Allowed gap symbols are the hyphen (-), and dot (.).


Please note that many programs that output this format tend to truncate the sequence names to a limited number of characters, for instance the first 15 characters. This can destroy the uniqueness of identifiers in your MSA.

Here is an example alignment in ClustalW format:

CLUSTAL W (1.83) multiple sequence alignment


AL031296.1/85969-86120      UCUCGUUGGUGAUAAGGAACAGCU

Stockholm 1.0 format

Here is an example alignment in Stockholm 1.0 format:


#=GF AC   RF01293
#=GF ID   ACA59
#=GF DE   Small nucleolar RNA ACA59
#=GF AU   Wilkinson A
#=GF SE   Predicted; WAR; Wilkinson A
#=GF SS   Predicted; WAR; Wilkinson A
#=GF GA   43.00
#=GF TC   44.90
#=GF NC   40.30
#=GF TP   Gene; snRNA; snoRNA; HACA-box;
#=GF BM   cmbuild -F CM SEED
#=GF CB   cmcalibrate --mpi CM
#=GF SM   cmsearch --cpu 4 --verbose --nohmmonly -E 1000 -Z 549862.597050 CM SEQDB
#=GF DR   snoRNABase; ACA59;
#=GF DR   SO; 0001263; ncRNA_gene;
#=GF DR   GO; 0006396; RNA processing;
#=GF DR   GO; 0005730; nucleolus;
#=GF RN   [1]
#=GF RM   15199136
#=GF RT   Human box H/ACA pseudouridylation guide RNA machinery.
#=GF RA   Kiss AM, Jady BE, Bertrand E, Kiss T
#=GF RL   Mol Cell Biol. 2004;24:5797-5807.
#=GF WK   Small_nucleolar_RNA
#=GF SQ   3

#=GC SS_cons               -----((((,<<<<<<<<<___________>>>>>>>>>,,,,<<<<<<<______>>>>>>>,,,,,))))::::::::::::
#=GC RF                    CUGCcccaCAaCacuuguGCCUCaGUUACcCauagguGuAGUGaGgGuggcAaUACccaCcCucgUUgGuggUaAGGAaCAgCU

See also…

WUSS notation for legal characters and their interpretation in the consensus secondary structure line SS_cons.

FASTA (Pearson) format


Sequence names must not contain whitespace characters. Otherwise, the parts after the first whitespace will be dropped. The only allowed gap character is the hyphen (-).

Here is an example alignment in FASTA format:


MAF format

The multiple alignment format (MAF) is usually used to store multiple alignments on DNA level between entire genomes. It consists of independent blocks of aligned sequences which are annotated by their genomic location. Consequently, an MAF formatted MSA file may contain multiple records. MAF files start with a line:


which is optionally extended by whitespace delimited key=value pairs. Lines starting with the character (#) are considered comments and usually ignored.

A MAF block starts with character (a) at the beginning of a line, optionally followed by whitespace delimited key=value pairs. The next lines start with character (s) and contain sequence information of the form:

s src start size strand srcSize sequence


  • src is the name of the sequence source

  • start is the start of the aligned region within the source (0-based)

  • size is the length of the aligned region without gap characters

  • strand is either (+) or (-), depicting the location of the aligned region relative to the source

  • srcSize is the size of the entire sequence source, e.g. the full chromosome

  • sequence is the aligned sequence including gaps depicted by the hyphen (-)

Here is an example alignment in MAF format (bluntly taken from the UCSC Genome browser website):

##maf version=1 scoring=tba.v8 
# tba.v8 (((human chimp) baboon) (mouse rat)) 
# multiz.v7
# maf_project.v5 _tba_right.maf3 mouse _tba_C
# single_cov2.v4 single_cov2 /dev/stdin
a score=23262.0     
s hg16.chr7    27578828 38 + 158545518 AAA-GGGAATGTTAACCAAATGA---ATTGTCTCTTACGGTG
s panTro1.chr6 28741140 38 + 161576975 AAA-GGGAATGTTAACCAAATGA---ATTGTCTCTTACGGTG
s baboon         116834 38 +   4622798 AAA-GGGAATGTTAACCAAATGA---GTTGTCTCTTATGGTG
s mm4.chr6     53215344 38 + 151104725 -AATGGGAATGTTAAGCAAACGA---ATTGTCTCTCAGTGTG
s rn3.chr4     81344243 40 + 187371129 -AA-GGGGATGCTAAGCCAATGAGTTGTTGTCTCTCAATGTG
a score=5062.0                    
s hg16.chr7    27699739 6 + 158545518 TAAAGA
s panTro1.chr6 28862317 6 + 161576975 TAAAGA
s baboon         241163 6 +   4622798 TAAAGA 
s mm4.chr6     53303881 6 + 151104725 TAAAGA
s rn3.chr4     81444246 6 + 187371129 taagga

a score=6636.0
s hg16.chr7    27707221 13 + 158545518 gcagctgaaaaca
s panTro1.chr6 28869787 13 + 161576975 gcagctgaaaaca
s baboon         249182 13 +   4622798 gcagctgaaaaca
s mm4.chr6     53310102 13 + 151104725 ACAGCTGAAAATA