source: trunk/GDE/CLUSTALW/clustalw.txt

README for Clustal W version 1.7  June 1997

             Clustal W version 1.7 Documentation

This file provides some notes on the latest changes, installation and usage
of the Clustal W multiple sequence alignment program.



Julie Thompson (Thompson@EMBL-Heidelberg.DE)
Toby Gibson    (Gibson@EMBL-Heidelberg.DE)

European Molecular Biology Laboratory
Meyerhofstrasse 1
D 69117 Heidelberg
Germany


Des Higgins (Higgins@ucc.ie)

University of County Cork
Cork
Ireland


Please e-mail bug reports/complaints/suggestions (polite if possible)
to Toby Gibson or Des Higgins.  



Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994)
CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment
through sequence weighting, positions-specific gap penalties and weight matrix
choice.  Nucleic Acids Research, 22:4673-4680.

--------------------------------------------------------------

What's New (June 1997) in Version 1.7 (since version 1.6).


1. The static arrays used by clustalw for storing the alignment data have been
replaced by dynamically allocated memory. There is now no limit on the number
or length of sequences which can be input.

2. The alignment of DNA sequences now offers a new hard-coded matrix, as well
as the identity matrix used previously. The new matrix is the default scoring
matrix used by the BESTFIT program of the GCG package 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 0.0.

3. The transition weight option for aligning nucleotide sequences has been
changed from an on/off toggle to a weight between 0 and 1.  A weight of zero
means that the transitions are scored as mismatches; a weight of 1 gives 
transitions the full 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.

4. The RSF sequence alignment file format used by GCG Version 9 can now be
read.

5. The clustal sequence alignment file format has been changed to allow
sequence names longer than 10 characters. The maximum length allowed is set in
clustalw.h by the statement:

        #define MAXNAMES        10

For the fasta format, the name is taken as the first string after the '>'
character, stopping at the first white space. (Previously, the first 10
characters were taken, replacing blanks by underscores).

6. The bootstrap values written in the phylip tree file format can be assigned
either to branches or nodes. The default is to write the values on the nodes,
as this can be read by several commonly-used tree display programs. But note
that this can lead to confusion if the tree is rooted and the bootstraps may
be better attached to the internal branches: Software developers should ensure
they can read the branch label format.

7. The sequence weighting used during sequence to profile alignments has been
changed. The tree weight is now multiplied by the percent identity of the
new sequence compared with the most closely related sequence in the profile.

8. The sequence weighting used during profile to profile alignments has been
changed. A guide tree is now built for each profile separately and the
sequence weights calculated from the two trees. The weights for each
sequence are then multiplied by the percent identity of the sequence compared
with the most closely related sequence in the opposite profile.

9. The adjustment of the Gap Opening and Gap Extension Penalties for sequences
of unequal length has been improved.

10. The default order of the sequences in the output alignment file has been
changed. Previously the default was to output the sequences in the same order
as the input file. Now the default is to use the order in which the sequences
were aligned (from the guide tree/dendrogram), thus automatically grouping
closely related sequences.

11. The option to 'Reset Gaps between alignments' has been switched off by
default.

12. The conservation line output in the clustal format alignment file has been
changed. Three characters are now used:

        '*' indicates positions which have a single, fully conserved residue

        ':' indicates that one of the following 'strong' groups is fully conserved:-

                 STA
                 NEQK
                 NHQK
                 NDEQ
                 QHRK
                 MILV
                 MILF
                 HY
                 FYW

        '.' indicates that one of the following 'weaker' groups is fully conserved:-

                 CSA
                 ATV
                 SAG
                 STNK
                 STPA
                 SGND
                 SNDEQK
                 NDEQHK
                 NEQHRK
                 FVLIM
                 HFY

These are all the positively scoring groups that occur in the Gonnet Pam250
matrix. The strong and weak groups are defined as strong score >0.5 and weak
score =<0.5 respectively.

13. A bug in the modification of the Myers and Miller alignment algorithm
for residue-specific gap penalites has been fixed. This occasionally caused
new gaps to be opened a few residues away from the optimal position.

14. The GCG/MSF input format no longer needs the word PILEUP on the first
line. Several versions can now be recognised:-
      1.  The word PILEUP as the first word in the file
      2.  The word !!AA_MULTIPLE_ALIGNMENT or !!NA_MULTIPLE_ALIGNMENT
          as the first word in the file
      3.  The characters MSF on the first line in the line, and the
          characters .. at the end of the line.

15. The standard command line separator for UNIX systems has been changed from
'/' to '-'. ie. to give options on the command line, you now type

     clustalw input.aln -gapopen=8.0

instead of

     clustalw input.aln /gapopen=8.0


                      ATTENTION SOFTWARE DEVELOPERS!!
                      -------------------------------

The CLUSTAL sequence alignment output format has been modified:

1. Names longer than 10 chars are now allowed. (The maximum is specified in
clustalw.h by '#define MAXNAMES'.)

2. The consensus line now consists of three characters: '*',':' and '.'. (Only
the '*' and '.' were previously used.)

3. An option (not the default) has been added, allowing the user to print out
sequence numbers at the end of each line of the alignment output.

4. Both RNA bases (U) and base ambiguities are now supported in nucleic acid
sequences. In the past, all characters (upper or lower case) other than
a,c,g,t or u were converted to N. Now the following characters are recognised 
and retained in the alignment output: ABCDGHKMNRSTUVWXY (upper or lower case).

5. A Blank line inadvertently added in the version 1.6 header has been taken
out again.


--------------------------------------------------------------

What's New (March 1996) in Version 1.6 (since version 1.5).


1) Improved handling of sequences of unequal length.  Previously, we
increased the gap extension penalties for both sequences if the two sequences
(or groups of previously aligned sequences) were of different lengths.  
Now, we increase the gap opening and extension penalties for the shorter 
sequence only.   This helps prevent short sequences being stretched out
along longer ones.

2) Added the "Gonnet" series of weight matrices (from Gaston Gonnet and 
co-workers at the ETH in Zurich).  Fixed a bug in the matrix
choice menu; now PAM matrices can be selected ok.

3) Added secondary structure/gap penalty masks.  These allow you to 
include, in an alignment, a position specific set of gap penalties.  
You can either set a gap opening penalty at each position or specify
the secondary strcuture (if protein; alpha helix, beta strand or loop)
and have gap penalties set automatically.   This, basically, is used to make 
gaps harder to open inside helices or strands.  

These masks are only used in the "profile alignment" menu.  They may be read in
as part of an alignment in a special format (see the on-line help for
details) or associated with each sequence, if the sequences are in Swiss Prot 
format and secondary structure information is given.   All of the mask 
parameters can be set from the profile alignment menu.  Basically, the
mask is made up of a series of numbers between 1 and 9, one per position.
The gap opening penalty at a position is calculated as the starting penalty
multipleied by the mask value at that site. 

4) Added command line options /profile and /sequences.
These allow uses to choose between normal profile alignment where the
two profiles (pre-existing alignments specified in the files
/profile1= and /profile2=) are merged/aligned with each other (/profile)
and the case where the individual sequences in /profile2 are aligned
sequentially with the alignment in /profile1 (/sequences).

5) Fixed bug in modified Myers and Miller algorithm - gap penalty score
was not always calculated properly for type 2 midpoints.  This is the core
alignment algorithm.

6) Only allows one output file format to be selected from command line
- ie. multiple output alignment files are not allowed.

7) Fixed 'bad calls to ckfree' error during calculation of phylip distance
matrix.

8) Fixed command line options /gapopen /gapext /type=protein /negative.

9) Allowed user to change command line separator on UNIX from '/' to '-'.
This allows unix users to use the more conventinal '-' symbol
for seperating command line options.  "/" can then be used in unix
file names on the command line.   The symbol that is used,
is specified in the file clustalw.h which must be edited if you 
wish to change it (and the program must then be recompiled).   Find the 
block of code in clustalw.h that corrsponds to the operating system you
are using.  These blocks are started by one of the following:

#ifdef VMS 
#elif MAC
#elif MSDOS
#elif UNIX

On the next line after each is the line:

#define COMMANDSEP '/'

Change this in the appropriate block of code (e.g. the UNIX block) to 

#define COMMANDSEP '-'

if you wish to use the "-" character as command seperator.
  

       
--------------------------------------------------------------

What's New (April 1995) in Version 1.5 (since version 1.3).

1) ported to MAC and PC.  These versions are quite slow unless you
have a nice beefy machine.  On a Power Mac or a Pentium box
it is nice and fast.  Two precompiled versions are supplied for Macs
(Power mac and old mac versions).

Mac:       1500 residues by 100 sequences
Power Mac  3000    "     "   "     "
PC         1500    "     "   "     "

2) alignment of new sequences to an alignment.  Fixed a serious bug
which assigned weights to the wrong sequences.  Now also, weights 
sequences according to distance from the incoming sequence.  The
new weights are: tree weights * similarity to incoming sequence.
The tree weights are the old weights that we derive from the tree
connecting all the sequences in the existing alignment.

3) for all platforms, output linelength = 60.

4) Bootstrap files (*.phb): the "final" node (arbitrary trichotomy
at the end of the neighbor-joining process) is labelled as 
TRICHOTOMY in the bootstrap output files.  This is to help
link bootstrap figures with nodes when you reroot the tree.

5) Command line /bootstrap option now more robust.

--------------------------------------------------------------
INTRODUCTION



This document gives some BRIEF notes about usage of the Clustal W
multiple alignment program for UNIX and VMS machines.  Clustal W
is a major update and rewrite of the Clustal V program which 
was described in:

Higgins, D.G., Bleasby, A.J. and Fuchs, R. (1992)
CLUSTAL V: improved software for multiple sequence alignment.
Computer Applications in the Biosciences (CABIOS), 8(2):189-191.

The main new features are a greatly improved (more sensitive)
multiple alignment procedure for proteins and improved support
for different file formats.  This software was described in:

Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994)
CLUSTAL W: improving the sensitivity of progressive multiple
sequence alignment through sequence weighting, position specific
gap penalties and weight matrix choice.
Nucleic Acids Research, 22(22):4673-4680.


The usage of Clustal W is largely the same as for
Clustal V details of which are described in clustalv.txt.  Details of the
new alignment algorithms are described in the manuscript by
Thompson et. al. above, an ascii/text version of which is included 
(clustalw.ms). This file lists some of the details not covered by either 
of the above documents.


There are brief notes on the following topics:

1) Installation for VMS and UNIX and MAC and PC
2) File input
3) file output
4) changes to the alignment algorithms
5) minor modifications to the phylogenetic tree and bootstrapping methods
6) summary of the command line usage.

-------------------------------------------------------------------

1) INSTALLATION    (for Unix, VAX/VMS, PC and MAC)



*****IMPORTANT*****
If you wish to recompile the program (or compile it for the first
time; you will have to do this with UNIX):
first check the file CLUSTALW.H which needs to be changed if you
move the code from between unix and vms machines.  At the top
of the file are four lines which define one of VMS, MSDOS, MAC or
UNIX to be 1.  All of these EXCEPT one must be commented out
using enclosed /* ... */.  
*******************


Unix
-----

Make files are supplied for unix machines.  The code was compiled and
tested using Decstation (Ultrix), SUN (Gnu C compiler/gcc), Silicon
Graphics (IRIX) and DEC/Alpha (OSF1).  We have not tested the code on any other
systems.  Just use makefile to make on most systems.  For Sun, you need to
have the Gnuc C (gcc) compiler installed ... use the file makefile.sun in this
case.  You make the program with:
make  (or make -f makefile.sun)

This produces the file clustalw which can be run by typing clustalw and
pressing return.  The help file is called clustalw_help


VMS
----

There is a small DCL command file (VMSLINK.COM) to compile and link the
code for VMS machines (vax or alpha).  This procedure just compiles the
source files and links using default settings.  Run it using:
$ @vmslink
This produces Clustalw.exe which can be run using the run command:
$ run clustalw

The intermediate object files can be deleted with:
$ del *.obj;

There is an extensive command line facility.  To use this, you must
create a symbol to run the program (and put this in your login.com file).
e.g.
$ clustalw :== $$drive:[dir.dir]clustalw
where $drive is the drive on which the executable file is stored (clustalw.exe)
and [dir.dir] is the full directory specification.  NOTE THE EXTRA DOLLAR SIGN.
Then the program can be run using the command:
$ clustalw


PC
__

We supply an executable file (Clustalw.exe) which will run using MSDOS.
It will also run under windows (as a DOS application) 
*** IF you have a maths coprocessor***.  If you do not have a maths chip 
(e.g. 80387), the program can only be run under MSDOS.  In the latter case, 
you must have the file EMU387.exe in the same directory as CLUSTALW.EXE.  
This file emulates a maths chip if you do not have one.  


We generated the executable file using gnu c for MSDOS. 
It will also compile (with about 10,000 warning messages)
using Microsoft C but we have not tested it and there appear to be problems
with the executable. 

You will need to use a "memory extender" to allow the program to get at more 
than 640kb of memory.



MAC
---

The code compiles for Power Mac and older macs using Metroworks Codewarrior
C compiler.  We supply 2 executable programs (one each for PowerMac and
older mac): ClustalwPPC and Clustalw68k).  These need up to
10mb of memory to run which needs to be adjusted with the Get Info (%I)
command from the Finder if you have problems.  Just double click the 
executable file name or icon and off you go (we hope).

As a special treat for Mac users, we supply an executable and brief readme
file for NJPLOT.   This is a really nice program by Manolo Gouy
(University of Lyon, France) that allows you to import the trees
made by Clustal W and display them/manipulate them.  It will properly
display the bootstrap figures from the *.phb files.  It can export the
trees in PICT format which can then be used by MacDraw for example.


-------------------------------------------------------------------------

2) FILE INPUT (sequences to be aligned)



The sequences must all be in one file (or two files for a "profile alignment")
in ONE of the following formats:

FASTA (Pearson), NBRF/PIR, EMBL/Swiss Prot, GDE, CLUSTAL, GCG/MSF, GCG9/RSF.

The program tries to "guess" which format is being used and whether
the sequences are nucleic acid (DNA/RNA) or amino acid (proteins).  The
format is recognised by the first characters in the file.  This is kind
of stupid/crude but works most of the time and it is difficult
to do reliably, any other way.


Format           First non blank word or character in the file.
...............................................................
FASTA            >
NBRF             >P1;  or >D1;
EMBL/SWISS       ID
GDE protein      % 
GDE nucleotide   # 
CLUSTAL          CLUSTAL (blocked multiple alignments)
GCG/MSF          PILEUP  or !!AA_MULTIPLE_ALIGNMENT or !!NA_MULTIPLE_ALIGNMENT
                 or MSF on the first line, and '..' at the end of line
GCG9/RSF         !!RICH_SEQUENCE

Note, that the only way of spotting that a file is MSF format is if
the word PILEUP appears at the very beginning of the file.  If you 
produce this format from software other than the GCG pileup program,
then you will have to insert the word PILEUP at the start of the file.
Similarly, if you use clustal format, the word CLUSTAL must appear first.

All of these formats can be used to read in AN EXISTING FULL ALIGNMENT.
With CLUSTAL format, this is just the same as the output format of this
program and Clustal V.  If you use PILEUP or CLUSTAL format, all sequences
must be the same length, INCLUDING GAPS ("-" in clustal format; "." in MSF).
With the other formats, sequences can be gapped with "-" characters.  If you
read in any gaps these are kept during any later alignments.  You can use
this facility to read in an alignment in order to calculate a phylogenetic
tree OR to output the same alignment in a different format (from the
output format options menu of the multiple alignment menu) e.g. read
in a GCG/MSF format alignment and output a PHYLIP format alignment. This is 
also useful to read in one reference alignment and to add one or more new 
sequences to it using the "profile alignment" facilities.

DNA vs. PROTEIN:  the program will count the number of A,C,G,T,U and N
charcters.  If 85% or more of the characters in a sequence are as above,
then DNA/RNA is assumed, protein otherwise.  

-------------------------------------------------------------------------


3) FILE OUTPUT 


        1) the alignments.

        In the multiple alignment and profile alignment menus, there is a menu
        item to control the output format(s).

        The alignment output format can be set to any (or all) of:
        CLUSTAL  (a self explanatory blocked alignment)
        NBRF/PIR (same as input format but with "-" characters for gaps)
        MSF      (the main GCG package multiple alignment format)
        PHYLIP   (Joe Felsenstein's phylogeny inference package.  Gaps are set to
                 "-" characters.  For some programs (e.g. PROTPARS/DNAPARS) these 
                 should be changed to "?" characters for unknown residues.
        GDE      (Used by Steven Smith's GDE package)

        You can also choose between having the sequences in the same order as in 
        the input file or writing them out in an order that more closely matches the 
        order used to carry out the multiple alignment.
        
        
        2) The trees.
        
        Believe it or not, we now use the New Hampshire (nested parentheses)
        format as default for our trees.  This format is compatible with e.g. the
        PHYLIP package.  If you want to view a tree, you can use the RETREE or 
        DRAWGRAM/DRAWTREE programs of PHYLIP.  This format is used for all our 
        trees, even the initial guide trees for deciding the order of multiple
        alignment.  The output trees from the phylogenetic tree menu can also be
        requested in our old verbose/cryptic format.  This may be more useful
        if, for example, you wish to see the bootstrap figures.  The bootstrap
        trees in the default New Hampshire format give the bootstrap figures
        as extra labels which can be viewed very easily using TREETOOL which is
        available as part of the GDE package.  TREETOOL is available from the
        RDP project by ftp from rdp.life.uiuc.edu.  
        
        The New Hampshire format is only useful if you have software to display or
        manipulate the trees.  The PHYLIP package is highly recommended if you intend
        to do much work with trees and includes programs for doing this.  If you do
        not have such software, request the trees in the older clustal format
        and see the documentation for Clustal V (clustalv.txt).  WE DO NOT PROVIDE
        ANY DIRECT MEANS FOR VIEWING TREES GRAPHICALLY.

-------------------------------------------------------------------------

4) THE ALIGNMENT ALGORITHMS


The basic algorithm is the same as for Clustal V and is described in some
detail in clustalv.txt.  The new modifications are described in detail in 
clustalw.ms.  Here we just list some notes to help answer some of the most
obvious questions.


Terminal Gaps

In the original Clustal V program, terminal gaps were penalised the same
as all other gaps.  This caused some ugly side effects e.g.

acgtacgtacgtacgt                              acgtacgtacgtacgt
a----cgtacgtacgt  gets the same score as      ----acgtacgtacgt

NOW, terminal gaps are free.  This is better on average and stops silly
effects like single residues jumping to the edge of the alignment.  However,
it is not perfect.  It does mean that if there should be a gap near the end 
of the alignment, the program may be reluctant to insert it i.e. 

cccccgggccccc                                              cccccgggccccc
ccccc---ccccc  may be considered worse (lower score) than  cccccccccc---

In the right hand case above, the terminal gap is free and may score higher
than the laft hand alignment.  This can be prevented by lowering the gap
opening and extension penalties.   It is difficult to get this right all the
time.  Please watch the ends of your alignments. 



Speed of the initial (pairwise) alignments (fast approximate/slow accurate)

By default, the initial pairwise alignments are now carried out using a full
dynamic programming algorithm.  This is more accurate than the older hash/
k-tuple based alignments (Wilbur and Lipman) but is MUCH slower.  On a fast
workstation you may not notice but on a slow box, the difference is extreme.
You can set the alignment method from the menus easily to the older, faster
method.



Delaying alignment of distant sequences

The user can set a cut off to delay the alignment of the most divergent
sequences in a data set until all other sequences have been aligned.  By 
default, this is set to 40% which means that if a sequence is less than 40%
identical to any other sequence, its alignment will be delayed.  



Iterative realignment/Reset gaps between alignments

By default, if you align a set of sequences a second time (e.g. with changed
gap penalties), the gaps from the first alignment are discarded.  You can
set this from the menus so that older gaps will be kept between alignments,
This can sometimes give better alignments by keeping the gaps (do not reset
them) and doing the full multiple alignment a second time.  Sometimes, the
alignment will converge on a better solution; sometimes the new alignment will
be the same as the first.  There can be a strange side effect: you can get
columns of nothing but gaps introduced.  

Any gaps that are read in from the input file are always kept, regardless 
of the setting of this switch.  If you read in a full multiple alignment, the "reset
gaps" switch has no effect.  The old gaps will remain and if you carry out 
a multiple alignment, any new gaps will be added in.  If you wish to carry out 
a full new alignment of a set of sequences that are already aligned in a file
you must input the sequences without gaps.



Profile alignment

By profile alignment, we simply mean the alignment of old alignments/sequences.
In this context, a profile is just an existing alignment (or even a set of 
unaligned sequences; see below).  This allows you to
read in an old alignment (in any of the allowed input formats) and align
one or more new sequences to it.  From the profile alignment menu, you
are allowed to read in 2 profiles.  Either profile can be a full alignment
OR a single sequence.  In the simplest mode, you simply align the two profiles
to each other. This is useful if you want to gradually build up a full
multiple alignment.  

A second option is to align the sequences from the second profile, one at
a time to the first profile.  This is done, taking the underlying tree between
the sequences into account.  This is useful if you have a set of new sequences
(not aligned) and you wish to add them all to an older alignment.

----------------------------------------------------------------------------

5) CHANGES TO THE PHYLOGENTIC TREE CALCULATIONS AND SOME HINTS.



IMPROVED DISTANCE CALCULATIONS FOR PROTEIN TREES


The phylogenetic trees in Clustal W (the real trees that you calculate
AFTER alignment; not the guide trees used to decide the branching order
for multiple alignment) use the Neighbor-Joining method of Saitou and
Nei based on a matrix of "distances" between all sequences.  These distances
can be corrected for "multiple hits".  This is normal practice when accurate
trees are needed.  This correction stretches distances (especially large ones)
to try to correct for the fact that OBSERVED distances (mean number of 
differences per site) greatly underestimate the actual number that happened
during evolution.  

In Clustal V we used a simple formula to convert an observed distance to one
that is corrected for multiple hits.  The observed distance is the mean number
of differences per site in an alignment (ignoring sites with a gap) and is
therefore always between 0.0 (for ientical sequences) an 1.0 (no residues the
same at any site).  These distances can be multiplied by 100 to give percent
difference values.  100 minus percent difference gives percent identity.
The formula we use to correct for multiple hits is from Motoo Kimura
(Kimura, M. The neutral Theory of Molecular Evolution, Camb.Univ.Press, 1983,
page 75) and is:

K = -Ln(1 - D - (D.D)/5)  where D is the observed distance and K is       
                              corrected distance.

This formula gives mean number of estimated substitutions per site and, in
contrast to D (the observed number), can be greater than 1 i.e. more than
one substitution per site, on average.  For example, if you observe 0.8
differences per site (80% difference; 20% identity), then the above formula
predicts that there have been 2.5 substitutions per site over the course 
of evolution since the 2 sequences diverged.  This can also be expressed in 
PAM units by multiplying by 100 (mean number of substitutions per 100 residues).
The PAM scale of evolution and its derivation/calculation comes from the
work of Margaret Dayhoff and co workers (the famous Dayhoff PAM series
of weight matrices also came from this work).  Dayhoff et al constructed
an elaborate model of protein evolution based on observed frequencies
of substitution between very closely related proteins.  Using this model,
they derived a table relating observed distances to predicted PAM distances.
Kimura's formula, above, is just a "curve fitting" approximation to this table.
It is very accurate in the range 0.75 > D > 0.0 but becomes increasingly
unaccurate at high D (>0.75) and fails completely at around D = 0.85.

To circumvent this problem, we calculated all the values for K corresponding
to D above 0.75 directly using the Dayhoff model and store these in an 
internal table, used by Clustal W.  This table is declared in the file dayhoff.h and
gives values of K for all D between 0.75 and 0.93 in intervals of 0.001 i.e.
for D = 0.750, 0.751, 0.752 ...... 0.929, 0.930.   For any observed D 
higher than 0.930, we arbitrarily set K to 10.0.  This sounds drastic but
with real sequences, distances of 0.93 (less than 7% identity) are rare.
If your data set includes sequences with this degree of divergence, you
will have great difficulty getting accurate trees by ANY method; the alignment
itself will be very difficult (to construct and to evaluate).

There are some important
things to note.  Firstly, this formula works well if your sequences are
of average amino acid composition and if the amino acids substitute according
to the original Dayhoff model.  In other cases, it may be misleading.  Secondly,
it is based only on observed percent distance i.e. it does not DIRECTLY
take conservative substitutions into account.  Thirdly, the error on the
estimated PAM distances may be VERY great for high distances; at very high
distance (e.g. over 85%) it may give largely arbitrary corrected distances.
In most cases, however, the correction is still worth using; the trees will
be more accurate and the branch lengths will be more realistic.  

A far more sophisticated distance correction based on a full Dayhoff
model which DOES take conservative substitutions and actual amino acid
composition into account, may be found in the PROTDIST program of the
PHYLIP package.  For serious tree makers, this program is highly recommended. 



TWO NOTES ON BOOTSTRAPPING...

When you use the BOOTSTRAP in Clustal W to estimate the reliability of parts
of a tree, many of the uncorrected distances may randomly exceed the arbitrary cut
off of 0.93 (sequences only 7% identical) if the sequences are distantly
related.  This will happen randomly i.e. even if none of the pairs of 
sequences are less than 7% identical, the bootstrap samples may contain pairs
of sequences that do exceed this cut off.
If this happens, you will be warned.  In practice, this can
happen with many data sets.  It is not a serious problem if it happens rarely.
If it does happen (you are warned when it happens and told how often the
problem occurs), you should consider removing the most distantly
related sequences and/or using the PHYLIP package instead.


A further problem arises in almost exactly the opposite situation: when
you bootstrap a data set which contains 3 or more sequences that are identical
or almost identical.  Here, the sets of identical sequences should be shown
as a multifurcation (several sequences joing at the same part of the tree).
Because the Neighbor-Joining method only gives strictly dichotomous trees
(never more than 2 sequences join at one time), this cannot be exactly 
represented.  In practice, this is NOT a problem as there will be some
internal branches of zero length seperating the sequences.  If you
display the tree with all branch lengths, you will still see a multifurcation.  
However, when you bootstrap
the tree, only the branching orders are stored and counted.  In the case
of multifurcations, the exact branching order is arbitrary but the program
will always get the same branching order, depending only on the input order
of the sequences.  In practice, this is only a problem in situations where
you have a set of sequences where all of them are VERY similar.  In this case,
you can find very high support for some groupings which will disappear if you
run the analysis with a different input order.  Again, the PHYLIP package
deals with this by offering a JUMBLE option to shuffle the input order
of your sequences between each bootstrap sample.  

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6) SUMMARY OF THE COMMAND LINE USAGE

Clustal W is designed to be run interactively.  However, there are many 
situations where it is convenient to run it from the command line, especially
if you wish to run it from another piece of software (e.g. SeqApp or GDE).
All parameters can be set from the command line by giving options after the
clustalw command. On UNIX options should be preceded by '-', all other systems
use the '/' character.

If anything is put on the command line, the program will (attempt to) carry
out whatever is requested and will exit.  If you wish to use the command
line to set some parameters and then go into interactive mode, use the
command line switch: interactive .... e.g.

clustalw -quicktree -interactive    on UNIX
or
clustalw /quicktree /interactive    on VMS,MAC and PC

will set the default initial alignment mode to fast/approximate and will then
go to the main menu.


To see a list of all the command line parameters, type: 

clustalw -options           on UNIX
or
clustalw /options           on VMS,MAC and PC

and you will see a list with no explanation.


To get (VERY BRIEF) help on command line usage, use the /HELP or /CHECK
(-help or -check on UNIX systems) options.  Otherwise, the command line
usage is self explanatory or is explained in clustalv.txt.  The defaults
for all parameters are set in the file param.h which can be changed easily 
(remember to recompile the program afterwards :-).

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