This is the on-line help file for CLUSTAL W, version 1.6, March 1996.

SEQUENCE INPUT: all sequences must be in 1 file, one after another. 6 formats are automatically recognised: NBRF/PIR, EMBL/SWISSPROT, Pearson (Fasta), Clustal (*.aln), GCG/MSF (Pileup) and GDE flat file. All non-alphabetic characters (spaces, digits, punctuation marks) are ignored except "-" which is used to indicate a GAP ("." in GCG/MSF).

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.

PROFILE ALIGNMENTS (menu item 3) are used to align 2 alignments. Use this to add a new sequence to an old alignment, or to use secondary structure to guide the alignment process. GAPS in the old alignments are indicated using the "-" character. PROFILES can be input in ANY of the allowed formats; just use "-" (or "." for MSF) for each gap position.

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 the word PileUp at the start of the file. If your msf files do not contain this word first, edit it in at the start of the first line.

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!

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.

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

RESET GAPS (menu item 6) 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 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 8 (format options) allows you to choose from 5 different alignment formats (CLUSTAL, GCG, NBRF/PIR, PHYLIP and GDE).

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. >20) 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. For DNA, an identity matrix is used.

1. only exactly matching fragments (k-tuples) are considered;
2. only the 'best' diagonals (the ones with most k-tuple matches) are used.

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.

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 penalised.
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. For DNA, the scoring system assigns a score of 3 for two identical bases and zero otherwise. The TOGGLE TRANSITIONS switch (menu item 3) gives transitions (A <--> G or C <--> T i.e. purine-purine or pyrimidine-pyrimidine substitutions) a score of 1; otherwise, these are scored as mismatches and get a score of zero. For distantly related DNA sequences, this switch might be better turned off; for closely related sequences it can be useful.
5. PROTEIN WEIGHT MATRIX leads to a new menu where you are offered a choice of weight matrices. The default is the BLOSUM series of matrices by Jorja and Steven Henikoff. 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 distance. Further help is offered in the weight matrix menu.

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. 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.
3. 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.
4. 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 meaningful.

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.

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 format is used by the GDE package of Steven Smith.

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

The profiles can be in any of the allowed input formats with "-" characters used to specify gaps (except for GCG/MSF where "." is used).

You have to specify the 2 profiles by choosing menu items 1 and 2 and giving 2 file names. Then Menu item 3 will align the 2 profiles to each other. Secondary structure masks in either profile can be used to guide the alignment.

Menu item 4 will take the sequences in the second profile and align them to the first profile, 1 at a time. This is useful to add some new sequences to an existing alignment, or to align a set of sequences to a known structure. In this case, the second profile need not be pre-aligned.

The alignment parameters can be set using menu items 5, 6 and 7. These are EXACTLY the same parameters as used by the general, automatic multiple alignment procedure. The general multiple alignment procedure is simply a series of profile alignments. Carrying out a series of profile alignments on larger and larger groups of sequences, allows you to manually build up a complete alignment, if necessary editing intermediate alignments.

SECONDARY STRUCTURE OPTIONS. Menu Option 0 allows you to set secondary structure parameters. If a solved structure is available, it can be used to guide the alignment by raising gap penalties within secondary structure elements, so that gaps will preferentially be inserted into unstructured surface loop regions. Alternatively, a user-specified gap penalty mask can be supplied for a similar purpose.

A gap penalty mask is a series of numbers between 1 and 9, one per position in the alignment. Each number specifies how much the gap opening penalty is to be raised by at that position (raised by multiplying the basic gap opening penalty by the number) i.e. a mask figure of 1 at a positiion means no change in gap opening penalty; a figure of 4 means that the gap opening penalty is four times greater at that position, making gaps 4 times harder to open.

The format for gap penalty masks and secondary structure masks is explained in the help under option 0 (secondary structure options).

Options 1 and 2 control whether the input secondary structure information or gap penalty masks will be used.

Option 3 controls whether the secondary structure and gap penalty masks should be included in the output alignment.

Options 4 and 5 provide the value for raising the gap penalty at core Alpha Helical (A) and Beta Strand (B) residues. In CLUSTAL format, capital residues denote the A and B core structure notation. Basic gap penalties are multiplied by the amount specified.

Option 6 provides the value for the gap penalty in Loops. By default this penalty is not raised. In CLUSTAL format, loops are specified by "." in the secondary structure notation.

Option 7 provides the value for setting the gap penalty at the ends of secondary structures. Ends of secondary structures are observed to grow and/or shrink in related structures. Therefore by default these are given intermediate values, lower than the core penalties. All secondary structure read in as lower case in CLUSTAL format gets the reduced terminal penalty.

Options 8 and 9 specify the range of structure termini for the intermediate penalties. In the alignment output, these are indicated as lower case. For Alpha Helices, by default, the range spans the end helical turn. For Beta Strands, the default range spans the end residue and the adjacent loop residue, since sequence conservation often extends beyond the actual H-bonded Beta Strand.

CLUSTAL W can read the masks from SWISS-PROT, CLUSTAL or GDE format input files. For many 3-D protein structures, secondary structure information is recorded in the feature tables of SWISS-PROT database entries. You should always check that the assignments are correct - some are quite inaccurate. CLUSTAL W looks for SWISS-PROT HELIX and STRAND assignments e.g.

FT   HELIX       100    115
FT   STRAND      118    119

!SS_HBA_HUMA    ..aaaAAAAAAAAAAaaa.aaaAAAAAAAAAAaaaaaaAaaa.........aaaAAAAAA
!GM_HBA_HUMA    112224444444444222122244444444442222224222111111111222444444
HBA_HUMA        VLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLSHGSAQVKGHGK

In GDE flat file format, the masks are specified as text and the names must begin with SS_ or GM_.

Either a structure or penalty mask or both may be used. If both are included in an alignment, the user will be asked which is to be used.

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 method used is the NJ (Neighbour Joining) method of Saitou and Nei. First you calculate distances (percent divergence) between all pairs of sequence from a multiple alignment; second you apply the NJ method to the distance matrix.
1. 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. 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.
2. CORRECT FOR MULTIPLE SUBSTITUTIONS? For small divergence (say <10%) this option makes no difference. For greater divergence, this option corrects for the fact that observed distances underestimate actual evolutionary dist- ances. 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.

   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.

3. 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).
4. BOOTSTRAPPING is a method for deriving confidence values for the groupings in a tree (first adapted for trees by Joe Felsenstein). It involves making N random samples of sites from the alignment (N should be LARGE, e.g. 500 - 1000); drawing N trees (1 from each sample) and counting how many times each grouping from the original tree occurs in the sample trees. You must supply a seed number for the random number generator. Different runs with the same seed will give the same answer. See the documentation for details.
5. OUTPUT FORMATS: three different formats are allowed. None of these displays the tree visually. You must make the tree yourself (on paper) using the results OR get the PHYLIP package and use the tree drawing facilities there. (Get the PHYLIP package anyway if you are interested in trees).

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 data base similarity (homology searches). The matrices used are: Blosum80, 62, 40 and 30.
2. MD (Jones, Taylor). These matrices were derived using almost the same procedure as the Dayhoff one (below) 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 MD40, 60, 250 and 350 matrices.
3. PAM (Dayhoff). These have been extremely widely used since the late '70s. We use the PAM 120, 160, 250 and 350 matrices.

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.

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.

-INFILE=file.ext                             :input sequences.
-PROFILE1=file.ext  and  -PROFILE2=file.ext  :profiles (old alignment).

                VERBS (do things)

-OPTIONS	    :list the command line paramters
-HELP  or -CHECK    :outline the command line params.
-ALIGN              :do full multiple alignment 
-PROFILE            :merge two alignments (PROFILE1 and 2) by profile alignment
-SEQUENCES          :sequentially add PROFILE2 sequences to PROFILE1 alignment
-TREE               :calculate NJ tree.
-BOOTSTRAP(=n)      :bootstrap a NJ tree (n= number of bootstraps; def. = 1000).

                PARAMETERS (set things)

***General settings:****
-INTERACTIVE :read command line, then enter normal interactive menus
-QUICKTREE   :use FAST algorithm for the alignment guide tree
-NEWTREE=    :file for new guide tree
-USETREE=    :file for old guide tree
-NEGATIVE    :protein alignment with negative values in matrix
-OUTFILE=    :sequence alignment file name
-OUTPUT=     :GCG, GDE, PHYLIP or PIR
-OUTORDER=   :INPUT or ALIGNED
-CASE        :LOWER or UPPER (for GDE output only)

***Fast Pairwise Alignments:***
-KTUP=n      :word size                  -TOPDIAGS=n  :number of best diags.
-WINDOW=n    :window around best diags.  -PAIRGAP=n   :gap penalty
-SCORE       :PERCENT or ABSOLUTE

***Slow Pairwise Alignments:***
-PWMATRIX=   :BLOSUM, PAM, MD, ID or filename
-PWGAPOPEN=f :gap opening penalty        -PWGAPEXT=f  :gap opening penalty

***Multiple Alignments:***
-MATRIX=     :BLOSUM, PAM, MD, ID or filename
-GAPOPEN=f   :gap opening penalty        -GAPEXT=f    :gap extension penalty
-ENDGAPS     :end gap separation pen. -GAPDIST=n   :gap separation pen. range
-NOPGAP      :Pascarella gaps off        -NOHGAP      :hydrophilic gaps off
-HGAPRESIDUES= :list hydrophilic res.    -MAXDIV=n    :% ident. for delay
-TYPE=       :PROTEIN or DNA             -TRANSITIONS :transitions NOT weighted.

***Trees:***                             -SEED=n    :seed number for bootstraps.
-KIMURA      :use Kimura's correction.   -TOSSGAPS  :ignore positions with gaps.

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 NODE.
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.
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).