CLUSTAL 2.1 Multiple Sequence Alignments


 The UPGMA algorithm has been added to allow faster tree construction. The user now
 has the choice of using Neighbour Joining or UPGMA. The default is still NJ, but the
 user can change this by setting the clustering parameter.

 A remove first iteration scheme has been added. This can be used to improve the final
 alignment or improve the alignment at each stage of the progressive alignment. During the 
 iteration step each sequence is removed in turn and realigned. If the resulting alignment 
 is better than the  previous alignment it is kept. This process is repeated until the score
 converges (the  score is not improved) or until the maximum number of iterations is 
 reached. The user can  iterate at each step of the progressive alignment by setting the 
 iteration parameter to  TREE or just on the final alignment by seting the iteration 
 parameter to ALIGNMENT. The default is no iteration. The maximum number of  iterations can 
 be set using the numiter parameter. The default number of iterations is 3.
 -NUMITER=n     :Maximum number of iterations to perform
 -FULLHELP      :Print out the complete help content

 -MAXSEQLEN=n   :Maximum allowed sequence length
 -QUIET         :Reduce console output to minimum
 -STATS=file    :Log some alignents statistics to file

>> HELP 1 <<             General help for CLUSTAL W (2.1)

Clustal W is a general purpose multiple alignment program for DNA or proteins.

SEQUENCE INPUT:  all sequences must be in 1 file, one after another.  
7 formats are automatically recognised: NBRF-PIR, EMBL-SWISSPROT, 
Pearson (Fasta), Clustal (*.aln), GCG-MSF (Pileup), GCG9-RSF and GDE flat file.
All non-alphabetic characters (spaces, digits, punctuation marks) are ignored
except "-" which is used to indicate a GAP ("." in MSF-RSF).  

To do a MULTIPLE ALIGNMENT on a set of sequences, use item 1 from this menu to 
INPUT them; go to menu item 2 to do the multiple alignment.

PHYLOGENETIC TREES (menu item 4) can be calculated from old alignments (read in
with "-" characters to indicate gaps) OR after a multiple alignment while the 
alignment is still in memory.

The program tries to automatically recognise the different file formats used
and to guess whether the sequences are amino acid or nucleotide.  This is not
always foolproof.

FASTA and NBRF-PIR formats are recognised by having a ">" as the first 
character in the file.  

EMBL-Swiss Prot formats are recognised by the letters
ID at the start of the file (the token for the entry name field).  

CLUSTAL format is recognised by the word CLUSTAL at the beginning of the file.

GCG-MSF format is recognised by one of the following:
       - the word PileUp at the start of the file. 
         at the start of the file.
       - the word MSF on the first line of the line, and the characters ..
         at the end of this line.

GCG-RSF format is recognised by the word !!RICH_SEQUENCE at the beginning of
the file.

If 85% or more of the characters in the sequence are from A,C,G,T,U or N, the
sequence will be assumed to be nucleotide.  This works in 97.3% of cases
but watch out!

>> HELP 2 <<             Help for multiple alignments

If you have already loaded sequences, use menu item 1 to do the complete
multiple alignment.  You will be prompted for 2 output files: 1 for the 
alignment itself; another to store a dendrogram that describes the similarity
of the sequences to each other.

Multiple alignments are carried out in 3 stages (automatically done from menu
item 1 ...Do complete multiple alignments now):

1) all sequences are compared to each other (pairwise alignments);

2) a dendrogram (like a phylogenetic tree) is constructed, describing the
approximate groupings of the sequences by similarity (stored in a file).

3) the final multiple alignment is carried out, using the dendrogram as a guide.

PAIRWISE ALIGNMENT parameters control the speed-sensitivity of the initial

MULTIPLE ALIGNMENT parameters control the gaps in the final multiple alignments.

RESET GAPS (menu item 7) will remove any new gaps introduced into the sequences
during multiple alignment if you wish to change the parameters and try again.
This only takes effect just before you do a second multiple alignment.  You
can make phylogenetic trees after alignment whether or not this is ON.
If you turn this OFF, the new gaps are kept even if you do a second multiple
alignment. This allows you to iterate the alignment gradually.  Sometimes, the 
alignment is improved by a second or third pass.

SCREEN DISPLAY (menu item 8) can be used to send the output alignments to the 
screen as well as to the output file.

You can skip the first stages (pairwise alignments; dendrogram) by using an
old dendrogram file (menu item 3); or you can just produce the dendrogram
with no final multiple alignment (menu item 2).

OUTPUT FORMAT: Menu item 9 (format options) allows you to choose from 6 
different alignment formats (CLUSTAL, GCG, NBRF-PIR, PHYLIP, GDE, NEXUS, and FASTA).  

>> HELP 3 <<             Help for pairwise alignment parameters

A distance is calculated between every pair of sequences and these are used to
construct the dendrogram which guides the final multiple alignment. The scores
are calculated from separate pairwise alignments. These can be calculated using
2 methods: dynamic programming (slow but accurate) or by the method of Wilbur
and Lipman (extremely fast but approximate). 

You can choose between the 2 alignment methods using menu option 8.  The
slow-accurate method is fine for short sequences but will be VERY SLOW for 
many (e.g. >100) long (e.g. >1000 residue) sequences.   

SLOW-ACCURATE alignment parameters:
	These parameters do not have any affect on the speed of the alignments. 
They are used to give initial alignments which are then rescored to give percent
identity scores.  These % scores are the ones which are displayed on the 
screen.  The scores are converted to distances for the trees.

1) Gap Open Penalty:      the penalty for opening a gap in the alignment.
2) Gap extension penalty: the penalty for extending a gap by 1 residue.
3) Protein weight matrix: the scoring table which describes the similarity
                          of each amino acid to each other.
4) DNA weight matrix:     the scores assigned to matches and mismatches 
                          (including IUB ambiguity codes).

FAST-APPROXIMATE alignment parameters:

These similarity scores are calculated from fast, approximate, global align-
ments, which are controlled by 4 parameters.   2 techniques are used to make
these alignments very fast: 1) only exactly matching fragments (k-tuples) are
considered; 2) only the 'best' diagonals (the ones with most k-tuple matches)
are used.

K-TUPLE SIZE:  This is the size of exactly matching fragment that is used. 
INCREASE for speed (max= 2 for proteins; 4 for DNA), DECREASE for sensitivity.
For longer sequences (e.g. >1000 residues) you may need to increase the default.

GAP PENALTY:   This is a penalty for each gap in the fast alignments.  It has
little affect on the speed or sensitivity except for extreme values.

TOP DIAGONALS: The number of k-tuple matches on each diagonal (in an imaginary
dot-matrix plot) is calculated.  Only the best ones (with most matches) are
used in the alignment.  This parameter specifies how many.  Decrease for speed;
increase for sensitivity.

WINDOW SIZE:  This is the number of diagonals around each of the 'best' 
diagonals that will be used.  Decrease for speed; increase for sensitivity.

>> HELP 4 <<             Help for multiple alignment parameters

These parameters control the final multiple alignment. This is the core of the
program and the details are complicated. To fully understand the use of the
parameters and the scoring system, you will have to refer to the documentation.

Each step in the final multiple alignment consists of aligning two alignments 
or sequences.  This is done progressively, following the branching order in 
the GUIDE TREE.  The basic parameters to control this are two gap penalties and
the scores for various identical-non-indentical residues.  

1) and 2) The GAP PENALTIES are set by menu items 1 and 2. These control the 
cost of opening up every new gap and the cost of every item in a gap. 
Increasing the gap opening penalty will make gaps less frequent. Increasing 
the gap extension penalty will make gaps shorter. Terminal gaps are not 

3) The DELAY DIVERGENT SEQUENCES switch delays the alignment of the most
distantly related sequences until after the most closely related sequences have 
been aligned.   The setting shows the percent identity level required to delay
the addition of a sequence; sequences that are less identical than this level
to any other sequences will be aligned later.

4) The TRANSITION WEIGHT gives transitions (A <--> G or C <--> T 
i.e. purine-purine or pyrimidine-pyrimidine substitutions) a weight between 0
and 1; a weight of zero means that the transitions are scored as mismatches,
while a weight of 1 gives the transitions the match score. For distantly related
DNA sequences, the weight should be near to zero; for closely related sequences
it can be useful to assign a higher score.

5) PROTEIN WEIGHT MATRIX leads to a new menu where you are offered a choice of
weight matrices. The default for proteins in version 1.8 is the PAM series 
derived by Gonnet and colleagues. Note, a series is used! The actual matrix
that is used depends on how similar the sequences to be aligned at this 
alignment step are. Different matrices work differently at each evolutionary

6) DNA WEIGHT MATRIX leads to a new menu where a single matrix (not a series)
can be selected. The default is the matrix used by BESTFIT for comparison of
nucleic acid sequences.

Further help is offered in the weight matrix menu.

7)  In the weight matrices, you can use negative as well as positive values if
you wish, although the matrix will be automatically adjusted to all positive
scores, unless the NEGATIVE MATRIX option is selected.

8) PROTEIN GAP PARAMETERS displays a menu allowing you to set some Gap Penalty
options which are only used in protein alignments.

>> HELP A <<             Help for protein gap parameters.

1) RESIDUE SPECIFIC PENALTIES are amino acid specific gap penalties that reduce
or increase the gap opening penalties at each position in the alignment or
sequence.  See the documentation for details.  As an example, positions that 
are rich in glycine are more likely to have an adjacent gap than positions that
are rich in valine.

2) 3) HYDROPHILIC GAP PENALTIES are used to increase the chances of a gap within
a run (5 or more residues) of hydrophilic amino acids; these are likely to
be loop or random coil regions where gaps are more common.  The residues that 
are "considered" to be hydrophilic are set by menu item 3.

4) GAP SEPARATION DISTANCE tries to decrease the chances of gaps being too
close to each other. Gaps that are less than this distance apart are penalised
more than other gaps. This does not prevent close gaps; it makes them less
frequent, promoting a block-like appearance of the alignment.

5) END GAP SEPARATION treats end gaps just like internal gaps for the purposes
of avoiding gaps that are too close (set by GAP SEPARATION DISTANCE above).
If you turn this off, end gaps will be ignored for this purpose.  This is
useful when you wish to align fragments where the end gaps are not biologically

>> HELP 5 <<             Help for output format options.

Several output formats are offered. You can choose any (or all 6 if you wish).  

CLUSTAL format output is a self explanatory alignment format.  It shows the
sequences aligned in blocks.  It can be read in again at a later date to
(for example) calculate a phylogenetic tree or add a new sequence with a 
profile alignment.

GCG output can be used by any of the GCG programs that can work on multiple
alignments (e.g. PRETTY, PROFILEMAKE, PLOTALIGN).  It is the same as the GCG
.msf format files (multiple sequence file); new in version 7 of GCG.

Fasta output cis widely used because of it's simplicity. Each sequence name is
preceeded by a '>'-sign. The sequence itself is printed out in the following lines

PHYLIP format output can be used for input to the PHYLIP package of Joe 
Felsenstein.  This is an extremely widely used package for doing every 
imaginable form of phylogenetic analysis (MUCH more than the the modest intro-
duction offered by this program).

NBRF-PIR:  this is the same as the standard PIR format with ONE ADDITION.  Gap
characters "-" are used to indicate the positions of gaps in the multiple 
alignment.  These files can be re-used as input in any part of clustal that
allows sequences (or alignments or profiles) to be read in.  

GDE:  this is the flat file format used by the GDE package of Steven Smith.

NEXUS: the format used by several phylogeny programs, including PAUP and

GDE OUTPUT CASE: sequences in GDE format may be written in either upper or
lower case.

CLUSTALW SEQUENCE NUMBERS: residue numbers may be added to the end of the
alignment lines in clustalw format.

OUTPUT ORDER is used to control the order of the sequences in the output
alignments.  By default, the order corresponds to the order in which the
sequences were aligned (from the guide tree-dendrogram), thus automatically
grouping closely related sequences. This switch can be used to set the order
to the same as the input file.

PARAMETER OUTPUT: This option allows you to save all your parameter settings
in a parameter file. This file can be used subsequently to rerun Clustal W
using the same parameters.

>> HELP 6 <<             Help for phylogenetic trees

1) Before calculating a tree, you must have an ALIGNMENT in memory. This can be
input in any format or you should have just carried out a full multiple
alignment and the alignment is still in memory. 

*************** Remember YOU MUST ALIGN THE SEQUENCES FIRST!!!! ***************

The methods used are NJ (Neighbour Joining) and UPGMA. First
you calculate distances (percent divergence) between all pairs of sequence from
a multiple alignment; second you apply the NJ or UPGMA method to the distance matrix.

2) EXCLUDE POSITIONS WITH GAPS? With this option, any alignment positions where
ANY of the sequences have a gap will be ignored. This means that 'like' will be
compared to 'like' in all distances, which is highly desirable. It also
automatically throws away the most ambiguous parts of the alignment, which are
concentrated around gaps (usually). The disadvantage is that you may throw away
much of the data if there are many gaps (which is why it is difficult for us to
make it the default).  

3) CORRECT FOR MULTIPLE SUBSTITUTIONS? For small divergence (say <10%) this
option makes no difference. For greater divergence, it corrects for the fact
that observed distances underestimate actual evolutionary distances. This is
because, as sequences diverge, more than one substitution will happen at many
sites. However, you only see one difference when you look at the present day
sequences. Therefore, this option has the effect of stretching branch lengths
in trees (especially long branches). The corrections used here (for DNA or
proteins) are both due to Motoo Kimura. See the documentation for details.  

Where possible, this option should be used. However, for VERY divergent
sequences, the distances cannot be reliably corrected. You will be warned if
this happens. Even if none of the distances in a data set exceed the reliable
threshold, if you bootstrap the data, some of the bootstrap distances may
randomly exceed the safe limit.  

4) To calculate a tree, use option 4 (DRAW TREE NOW). This gives an UNROOTED
tree and all branch lengths. The root of the tree can only be inferred by
using an outgroup (a sequence that you are certain branches at the outside
of the tree .... certain on biological grounds) OR if you assume a degree
of constancy in the 'molecular clock', you can place the root in the 'middle'
of the tree (roughly equidistant from all tips).

By default, the bootstrap values are correctly placed on the tree branches of
the phylip format output tree. The toggle allows them to be placed on the
nodes, which is incorrect, but some display packages (e.g. TreeTool, TreeView
and Phylowin) only support node labelling but not branch labelling. Care
should be taken to note which branches and labels go together.

6) OUTPUT FORMATS: four different formats are allowed. None of these displays
the tree visually. Useful display programs accepting PHYLIP format include
NJplot (from Manolo Gouy and supplied with Clustal W), TreeView (Mac-PC), and
PHYLIP itself - OR get the PHYLIP package and use the tree drawing facilities
there. (Get the PHYLIP package anyway if you are interested in trees). The
NEXUS format can be read into PAUP or MacClade.

>> HELP 7 <<             Help for choosing a weight matrix

For protein alignments, you use a weight matrix to determine the similarity of
non-identical amino acids.  For example, Tyr aligned with Phe is usually judged 
to be 'better' than Tyr aligned with Pro.

There are three 'in-built' series of weight matrices offered. Each consists of
several matrices which work differently at different evolutionary distances. To
see the exact details, read the documentation. Crudely, we store several
matrices in memory, spanning the full range of amino acid distance (from almost
identical sequences to highly divergent ones). For very similar sequences, it
is best to use a strict weight matrix which only gives a high score to
identities and the most favoured conservative substitutions. For more divergent
sequences, it is appropriate to use "softer" matrices which give a high score
to many other frequent substitutions.

1) BLOSUM (Henikoff). These matrices appear to be the best available for 
carrying out database similarity (homology searches). The matrices used are:
Blosum 80, 62, 45 and 30. (BLOSUM was the default in earlier Clustal W

2) PAM (Dayhoff). These have been extremely widely used since the late '70s.
We use the PAM 20, 60, 120 and 350 matrices.

3) GONNET. These matrices were derived using almost the same procedure as the
Dayhoff one (above) but are much more up to date and are based on a far larger
data set. They appear to be more sensitive than the Dayhoff series. We use the
GONNET 80, 120, 160, 250 and 350 matrices. This series is the default for
Clustal W version 1.8.

We also supply an identity matrix which gives a score of 1.0 to two identical 
amino acids and a score of zero otherwise. This matrix is not very useful.
Alternatively, you can read in your own (just one matrix, not a series).

A new matrix can be read from a file on disk, if the filename consists only
of lower case characters. The values in the new weight matrix must be integers
and the scores should be similarities. You can use negative as well as positive
values if you wish, although the matrix will be automatically adjusted to all
positive scores.

For DNA, a single matrix (not a series) is used. Two hard-coded matrices are 

1) IUB. This is the default scoring matrix used by BESTFIT for the comparison
of nucleic acid sequences. X's and N's are treated as matches to any IUB
ambiguity symbol. All matches score 1.9; all mismatches for IUB symbols score 0.
2) CLUSTALW(1.6). The previous system used by Clustal W, in which matches score
1.0 and mismatches score 0. All matches for IUB symbols also score 0.

INPUT FORMAT  The format used for a new matrix is the same as the BLAST program.
Any lines beginning with a # character are assumed to be comments. The first
non-comment line should contain a list of amino acids in any order, using the
1 letter code, followed by a * character. This should be followed by a square
matrix of integer scores, with one row and one column for each amino acid. The
last row and column of the matrix (corresponding to the * character) contain
the minimum score over the whole matrix.

>> HELP 8 <<             Help for command line parameters

                DATA (sequences)

-INFILE=file.ext                             :input sequences.

                VERBS (do things)

-OPTIONS            :list the command line parameters
-HELP  or -CHECK    :outline the command line params.
-FULLHELP           :output full help content.
-ALIGN              :do full multiple alignment.
-TREE               :calculate NJ tree.
-PIM                :output percent identity matrix (while calculating the tree)
-BOOTSTRAP(=n)      :bootstrap a NJ tree (n= number of bootstraps; def. = 1000).
-CONVERT            :output the input sequences in a different file format.

                PARAMETERS (set things)

***General settings:****
-INTERACTIVE :read command line, then enter normal interactive menus
-QUICKTREE   :use FAST algorithm for the alignment guide tree
-TYPE=       :PROTEIN or DNA sequences
-NEGATIVE    :protein alignment with negative values in matrix
-OUTFILE=    :sequence alignment file name
-CASE        :LOWER or UPPER (for GDE output only)
-SEQNOS=     :OFF or ON (for Clustal output only)
-SEQNO_RANGE=:OFF or ON (NEW: for all output formats)
-RANGE=m,n   :sequence range to write starting m to m+n
-MAXSEQLEN=n :maximum allowed input sequence length
-QUIET       :Reduce console output to minimum
-STATS=      :Log some alignents statistics to file

***Fast Pairwise Alignments:***
-KTUPLE=n    :word size
-TOPDIAGS=n  :number of best diags.
-WINDOW=n    :window around best diags.
-PAIRGAP=n   :gap penalty

***Slow Pairwise Alignments:***
-PWMATRIX=    :Protein weight matrix=BLOSUM, PAM, GONNET, ID or filename
-PWDNAMATRIX= :DNA weight matrix=IUB, CLUSTALW or filename
-PWGAPOPEN=f  :gap opening penalty        
-PWGAPEXT=f   :gap extension penalty

***Multiple Alignments:***
-NEWTREE=      :file for new guide tree
-USETREE=      :file for old guide tree
-MATRIX=       :Protein weight matrix=BLOSUM, PAM, GONNET, ID or filename
-DNAMATRIX=    :DNA weight matrix=IUB, CLUSTALW or filename
-GAPOPEN=f     :gap opening penalty        
-GAPEXT=f      :gap extension penalty
-ENDGAPS       :no end gap separation pen. 
-GAPDIST=n     :gap separation pen. range
-NOPGAP        :residue-specific gaps off  
-NOHGAP        :hydrophilic gaps off
-HGAPRESIDUES= :list hydrophilic res.    
-MAXDIV=n      :% ident. for delay
-TYPE=         :PROTEIN or DNA
-TRANSWEIGHT=f :transitions weighting
-NUMITER=n     :maximum number of iterations to perform
-NOWEIGHTS     :disable sequence weighting

-OUTPUTTREE=nj OR phylip OR dist OR nexus
-SEED=n        :seed number for bootstraps.
-KIMURA        :use Kimura's correction.   
-TOSSGAPS      :ignore positions with gaps.
-BOOTLABELS=node OR branch :position of bootstrap values in tree display

>> HELP 9 <<             Help for tree output format options

Four output formats are offered: 1) Clustal, 2) Phylip, 3) Just the distances
4) Nexus

None of these formats displays the results graphically. Many packages can
display trees in the the PHYLIP format 2) below. It can also be imported into
the PHYLIP programs RETREE, DRAWTREE and DRAWGRAM for graphical display. 
NEXUS format trees can be read by PAUP and MacClade.

1) Clustal format output. 
This format is verbose and lists all of the distances between the sequences and
the number of alignment positions used for each. The tree is described at the
end of the file. It lists the sequences that are joined at each alignment step
and the branch lengths. After two sequences are joined, it is referred to later
as a NODE. The number of a NODE is the number of the lowest sequence in that

2) Phylip format output.
This format is the New Hampshire format, used by many phylogenetic analysis
packages. It consists of a series of nested parentheses, describing the
branching order, with the sequence names and branch lengths. It can be used by
the RETREE, DRAWGRAM and DRAWTREE programs of the PHYLIP package to see the
trees graphically. This is the same format used during multiple alignment for
the guide trees. 

Use this format with NJplot (Manolo Gouy), supplied with Clustal W. Some other
packages that can read and display New Hampshire format are TreeView (Mac/PC),
TreeTool (UNIX), and Phylowin.

3) The distances only.
This format just outputs a matrix of all the pairwise distances in a format
that can be used by the Phylip package. It used to be useful when one could not
produce distances from protein sequences in the Phylip package but is now
redundant (Protdist of Phylip 3.5 now does this).

4) NEXUS FORMAT TREE. This format is used by several popular phylogeny programs,
including PAUP and MacClade. The format is described fully in:
Maddison, D. R., D. L. Swofford and W. P. Maddison.  1997.
NEXUS: an extensible file format for systematic information.
Systematic Biology 46:590-621.

By default, the bootstrap values are placed on the nodes of the phylip format
output tree. This is inaccurate as the bootstrap values should be associated
with the tree branches and not the nodes. However, this format can be read and
displayed by TreeTool, TreeView and Phylowin. An option is available to
correctly place the bootstrap values on the branches with which they are