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
CLUSTAL W
or
CLUSTALW
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.
Here is an example alignment in ClustalW format:
CLUSTAL W (1.83) multiple sequence alignment AL031296.1/85969-86120 CUGCCUCACAACGUUUGUGCCUCAGUUACCCGUAGAUGUAGUGAGGGUAACAAUACUUAC AANU01225121.1/438-603 CUGCCUCACAACAUUUGUGCCUCAGUUACUCAUAGAUGUAGUGAGGGUGACAAUACUUAC AAWR02037329.1/29294-29150 ---CUCGACACCACU---GCCUCGGUUACCCAUCGGUGCAGUGCGGGUAGUAGUACCAAU AL031296.1/85969-86120 UCUCGUUGGUGAUAAGGAACAGCU AANU01225121.1/438-603 UCUCGUUGGUGAUAAGGAACAGCU AAWR02037329.1/29294-29150 GCUAAUUAGUUGUGAGGACCAACU
Here is an example alignment in Stockholm 1.0 format:
# STOCKHOLM 1.0 #=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 AL031296.1/85969-86120 CUGCCUCACAACGUUUGUGCCUCAGUUACCCGUAGAUGUAGUGAGGGUAACAAUACUUACUCUCGUUGGUGAUAAGGAACAGCU AANU01225121.1/438-603 CUGCCUCACAACAUUUGUGCCUCAGUUACUCAUAGAUGUAGUGAGGGUGACAAUACUUACUCUCGUUGGUGAUAAGGAACAGCU AAWR02037329.1/29294-29150 ---CUCGACACCACU---GCCUCGGUUACCCAUCGGUGCAGUGCGGGUAGUAGUACCAAUGCUAAUUAGUUGUGAGGACCAACU #=GC SS_cons -----((((,<<<<<<<<<___________>>>>>>>>>,,,,<<<<<<<______>>>>>>>,,,,,)))):::::::::::: #=GC RF CUGCcccaCAaCacuuguGCCUCaGUUACcCauagguGuAGUGaGgGuggcAaUACccaCcCucgUUgGuggUaAGGAaCAgCU //
Here is an example alignment in FASTA format:
>AL031296.1/85969-86120 CUGCCUCACAACGUUUGUGCCUCAGUUACCCGUAGAUGUAGUGAGGGUAACAAUACUUAC UCUCGUUGGUGAUAAGGAACAGCU >AANU01225121.1/438-603 CUGCCUCACAACAUUUGUGCCUCAGUUACUCAUAGAUGUAGUGAGGGUGACAAUACUUAC UCUCGUUGGUGAUAAGGAACAGCU >AAWR02037329.1/29294-29150 ---CUCGACACCACU---GCCUCGGUUACCCAUCGGUGCAGUGCGGGUAGUAGUACCAAU GCUAAUUAGUUGUGAGGACCAACU
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
##maf
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
where
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
The RNAlib and many programs of the ViennaRNA Package can parse and apply data from so-called command files. These commands may refer to structure constraints or even extensions of the RNA folding grammar (such as Unstructured domains ). Commands are given as a line of whitespace delimited data fields. The syntax we use extends the constraint definitions used in the mfold / UNAfold software, where each line begins with a command character followed by a set of positions.
However, we introduce several new commands, and allow for an optional loop type context specifier in form of a sequence of characters, and an orientation flag that enables one to force a nucleotide to pair upstream, or downstream.
The following set of commands is recognized:
F
:math:` ldots ` ForceP
:math:` ldots ` ProhibitC
:math:` ldots ` Conflicts/Context dependencyA
:math:` ldots ` Allow (for non-canonical pairs)E
:math:` ldots ` Soft constraints for unpaired position(s), or base pair(s)UD
:math:` ldots ` Add ligand binding using the Unstructured domains featureThe optional loop type context specifier ```` [LOOP] may be a combination of the following:
E
:math:` ldots ` Exterior loopH
:math:` ldots ` Hairpin loopI
:math:` ldots ` Interior loopM
:math:` ldots ` Multibranch loopA
:math:` ldots ` All loopsFor structure constraints, we additionally allow one to address base pairs enclosed by a particular kind of loop, which results in the specifier ```` [WHERE] which consists of ```` [LOOP] plus the following character:
i
:math:` ldots ` enclosed pair of an Interior loopm
:math:` ldots ` enclosed pair of a Multibranch loopIf no ```` [LOOP] or ```` [WHERE] flags are set, all contexts are considered (equivalent to A
)
For particular nucleotides that are forced to pair, the following ```` [ORIENTATION] flags may be used:
U
:math:` ldots ` UpstreamD
:math:` ldots ` DownstreamIf no ```` [ORIENTATION] flag is set, both directions are considered.
Sequence positions of nucleotides/base pairs are :math:` 1- ` based and consist of three positions :math:` i ` , :math:` j ` , and :math:` k ` . Alternativly, four positions may be provided as a pair of two position ranges :math:` [i:j] ` , and :math:` [k:l] ` using the ‘-‘ sign as delimiter within each range, i.e. :math:` i-j ` , and :math:` k-l ` .
Below are resulting general cases that are considered valid constraints:
“Forcing a range of nucleotide positions to be paired” :
Syntax:
F i 0 k [WHERE] [ORIENTATION]
Description:
Enforces the set of :math:` k ` consecutive nucleotides starting at position :math:` i ` to be paired. The optional loop type specifier ```` [WHERE] allows to force them to appear as closing/enclosed pairs of certain types of loops.
“Forcing a set of consecutive base pairs to form” :
Syntax:
F i j k [WHERE]
Description:
Enforces the base pairs :math:` (i,j), ldots, (i+(k-1), j-(k-1)) ` to form. The optional loop type specifier ```` [WHERE] allows to specify in which loop context the base pair must appear.
“Prohibiting a range of nucleotide positions to be paired” :
Syntax:
P i 0 k [WHERE]
Description:
Prohibit a set of :math:` k ` consecutive nucleotides to participate in base pairing, i.e. make these positions unpaired. The optional loop type specifier ```` [WHERE] allows to force the nucleotides to appear within the loop of specific types.
“Probibiting a set of consecutive base pairs to form” :
Syntax:
P i j k [WHERE]
Description:
Probibit the base pairs :math:` (i,j), ldots, (i+(k-1), j-(k-1)) ` to form. The optional loop type specifier ```` [WHERE] allows to specify the type of loop they are disallowed to be the closing or an enclosed pair of.
“Prohibiting two ranges of nucleotides to pair with each other” :
Syntax:
P i-j k-l [WHERE]
Description:
Prohibit any nucleotide :math:` p in [i:j] ` to pair with any other nucleotide :math:` q in [k:l] ` . The optional loop type specifier ```` [WHERE] allows to specify the type of loop they are disallowed to be the closing or an enclosed pair of.
“Enforce a loop context for a range of nucleotide positions” :
Syntax:
C i 0 k [WHERE]
Description:
This command enforces nucleotides to be unpaired similar to prohibiting nucleotides to be paired, as described above. It too marks the corresponding nucleotides to be unpaired, however, the ```` [WHERE] flag can be used to enforce specfic loop types the nucleotides must appear in.
“Remove pairs that conflict with a set of consecutive base pairs” :
Syntax:
C i j k
Description:
Remove all base pairs that conflict with a set of consecutive base pairs :math:` (i,j), ldots, (i+(k-1), j-(k-1)) ` . Two base pairs :math:` (i,j) ` and :math:` (p,q) ` conflict with each other if :math:` i < p < j < q ` , or :math:` p < i < q < j ` .
“Allow a set of consecutive (non-canonical) base pairs to form” :
Syntax:
A i j k [WHERE]
Description:
This command enables the formation of the consecutive base pairs :math:` (i,j), ldots, (i+(k-1), j-(k-1)) ` , no matter if they are canonical , or non-canonical . In contrast to the above F
and W
commands, which remove conflicting base pairs, the A
command does not. Therefore, it may be used to allow non-canoncial base pair interactions. Since the RNAlib does not contain free energy contributions :math:` E_{ij} ` for non-canonical base pairs :math:` (i,j) ` , they are scored as the maximum of similar, known contributions. In terms of a Nussinov like scoring function the free energy of non-canonical base pairs is therefore estimated as
The optional loop type specifier ```` [WHERE] allows to specify in which loop context the base pair may appear.
“Apply pseudo free energy to a range of unpaired nucleotide positions” :
Syntax:
E i 0 k e
Description:
Use this command to apply a pseudo free energy of :math:` e ` to the set of :math:` k ` consecutive nucleotides, starting at position :math:` i ` . The pseudo free energy is applied only if these nucleotides are considered unpaired in the recursions, or evaluations, and is expected to be given in :math:` kcal / mol ` .
“Apply pseudo free energy to a set of consecutive base pairs” :
Syntax
E i j k e
Use this command to apply a pseudo free energy of :math:` e ` to the set of base pairs :math:` (i,j), ldots, (i+(k-1), j-(k-1)) ` . Energies are expected to be given in :math:` kcal / mol ` .
“Add ligand binding to unpaired motif (a.k.a. unstructured domains)” :
Syntax:
UD m e [LOOP]
Description:
Add ligand binding to unpaired sequence motif :math:` m ` (given in IUPAC format, capital letters) with binding energy :math:` e ` in particular loop type(s).
Example:
UD AAA -5.0 A
The above example applies a binding free energy of :math:` -5 kcal/mol ` for a motif AAA that may be present in all loop types.
Note
Sequence names and the sequences must not contain whitespace characters! Allowed gap symbols are the hyphen ** (“-“), and dot ** (“.”).
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 ** (“-“).
Warning
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.
See also:
Washington University Secondary Structure (WUSS) notation on legal characters for the consensus secondary structure line SS_cons and their interpretation