clustalw_help

This file has been automatically converted from the original documentation for easy use inside the ARB help system. Differences compared with the original documentation are unintentionally caused by the conversion process. In doubt please refer to the original documentation!

This is the on-line help file for CLUSTAL W ( version 1.83).

It should be named or defined as: clustalw_help except with MSDOS in which case it should be named CLUSTALW.HLP

For full details of usage and algorithms, please read the CLUSTALW.DOC file.

Toby  Gibson                         EMBL, Heidelberg, Germany.
Des   Higgins                        UCC, Cork, Ireland.
Julie Thompson                       IGBMC, Strasbourg, France.

Fasta output ===========

Write/Read sequence with range specified. The command line syntax
 for range specification is flexible. You can use one of the following
 syntax.

Range and range numbers. =======================

Include range numbers in the ouput.

The sequence range will be appended as to the names of the sequence.

PIM: Percentage Identity Matrix ===============================

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.

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-RSF) 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 one of the following:

• the word PileUp at the start of the file.
• the word !!AA_MULTIPLE_ALIGNMENT or !!NA_MULTIPLE_ALIGNMENT 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!

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):

PAIRWISE ALIGNMENT parameters control the speed-sensitivity of the initial alignments.

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).

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.

FAST-APPROXIMATE alignment parameters:

These similarity scores are calculated from fast, approximate, global alignments, 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.

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-identical residues.

Six 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.

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 introduction 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 MacClade.

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.

By PROFILE ALIGNMENT, we mean alignment using existing alignments. Profile alignments allow you to store alignments of your favourite sequences and add new sequences to them in small bunches at a time. A profile is simply an alignment of one or more sequences (e.g. an alignment output file from CLUSTAL W). Each input can be a single sequence. One or both sets of input sequences may include secondary structure assignments or gap penalty masks to guide the alignment.

The profiles can be in any of the allowed input formats with "-" characters used to specify gaps (except for MSF-RSF 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 would 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 2D 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 loops. Alternatively, a user-specified gap penalty mask can be supplied directly.

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 at that position (raised by multiplying the basic gap opening penalty by the number) i.e. a mask figure of 1 at a position 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).

The use of secondary structure-based penalties has been shown to improve the accuracy of multiple alignment. Therefore CLUSTAL W now allows gap penalty masks to be supplied with the input sequences. The masks work by raising gap penalties in specified regions (typically secondary structure elements) so that gaps are preferentially opened in the less well conserved regions (typically surface loops).

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. The 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

The structure and penalty masks can also be read from CLUSTAL alignment format as comment lines beginning "!SS_" or "!GM_" e.g.

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

Note that the mask itself is a set of numbers between 1 and 9 each of which is assigned to the residue(s) in the same column below.

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.

   The options in this menu let you choose whether or not to include the masks
in the CLUSTAL W output alignments. Showing both is useful for understanding
how the masks work. The secondary structure information is itself very useful
in judging the alignment quality and in seeing how residue conservation
patterns vary with secondary structure.

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.

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 available:

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.

DATA (sequences)

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

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

***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
-OUTPUT=     :GCG, GDE, PHYLIP, PIR or NEXUS
-OUTORDER=   :INPUT or ALIGNED
-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.

***Fast Pairwise Alignments:***
-KTUPLE=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=    :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 opening 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

***Profile Alignments:***
-PROFILE      :Merge two alignments by profile alignment
-NEWTREE1=    :file for new guide tree for profile1
-NEWTREE2=    :file for new guide tree for profile2
-USETREE1=    :file for old guide tree for profile1
-USETREE2=    :file for old guide tree for profile2

***Sequence to Profile Alignments:***
-SEQUENCES   :Sequentially add profile2 sequences to profile1 alignment
-NEWTREE=    :file for new guide tree
-USETREE=    :file for old guide tree

***Structure Alignments:***
-NOSECSTR1     :do not use secondary structure-gap penalty mask for profile 1
-NOSECSTR2     :do not use secondary structure-gap penalty mask for profile 2
-SECSTROUT=STRUCTURE or MASK or BOTH or NONE   :output in alignment file
-HELIXGAP=n    :gap penalty for helix core residues
-STRANDGAP=n   :gap penalty for strand core residues
-LOOPGAP=n     :gap penalty for loop regions
-TERMINALGAP=n :gap penalty for structure termini
-HELIXENDIN=n  :number of residues inside helix to be treated as terminal
-HELIXENDOUT=n :number of residues outside helix to be treated as terminal
-STRANDENDIN=n :number of residues inside strand to be treated as terminal
-STRANDENDOUT=n:number of residues outside strand to be treated as terminal

***Trees:***
-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

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.