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               CLUSTAL W Multiple Sequence Alignment Program
                        (version 1.83, Feb 2003)

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Please send bug reports, comments etc. to one of:-
	gibson@embl-heidelberg.de
	thompson@igbmc.u-strasbg.fr
	d.higgins@ucc.ie


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                  POLICY ON COMMERCIAL DISTRIBUTION OF CLUSTAL W

Clustal W is freely available to the user community. However, Clustal W is
increasingly being distributed as part of commercial sequence analysis
packages. To help us safeguard future maintenance and development, commercial
distributors of Clustal W must take out a NON-EXCLUSIVE LICENCE. Anyone
wishing to commercially distribute version 1.81 of Clustal W should contact the
authors unless they have previously taken out a licence.

******************************************************************************

Clustal W is written in ANSI-C and can be run on any machine with an ANSI-C
compiler. Executables are provided for several major platforms. 

Changes since CLUSTAL X Version 1.82
------------------------------------

1. The FASTA format has been added to the list of alignment output options.

2. It is now possible to save the residue ranges (appended after the sequence
names) when saving a specified range of the alignment.

3. The efficiency of  the neighour-joining algorithm has been improved. This
work was done by Tadashi Koike at the Center for Information Biology and DNA Data
Bank of Japan and FUJITSU Limited.

Some example speedups are given below : (timings on a SPARC64 CPU)

No. of sequences        original NJ     new NJ
     200                0' 12"          0.1"
     500                9' 19"          1.4"
     1000               XXXX            0' 31"

Changes since version 1.8 
--------------------------

1. ClustalW now returns error codes for some common errors when exiting. This
may be useful for people who run clustalw automatically from within a script.
Error codes are: 
	1	bad command line option
	2	cannot open sequence file
	3	wrong format in sequence file
	4	sequence file contains only 1 sequence (for multiple alignments)

2. Alignments can now be saved in Nexus format, for compatibility with PAUP, 
MacClade etc. For a description of the Nexus format, see:
Maddison, D. R., D. L. Swofford and W. P. Maddison.  1997.
NEXUS: an extensible file format for systematic information.
Systematic Biology 46:590-621.

3. Phylogenetic trees can also be saved in nexus format.

4. A ClustalW icon has been designed for MAC and PC systems.


Changes since version 1.74 
--------------------------

1. Some work has been done to automatically select the optimal parameters
depending on the set of sequences to be aligned. The Gonnet series of residue
comparison matrices are now used by default. The Blosum series remains as an
option. The default gap extension penalty for proteins has been changed to 0.2
(was 0.05).The 'delay divergent sequences' option has been changed to 30%
residue identity (was 40%).

2. The default parameters used when the 'Negative matrix' option is selected
have been optimised. This option may help when the sequences to be aligned are
not superposable over their whole lengths (e.g. in the presence of N/C terminal
extensions).

3. A bug in the calculation of phylogenetic trees for 2 sequences has been
fixed.

4. A command line option has been added to turn off the sequence weighting
calculation.

5. The phylogenetic tree calculation now ignores any ambiguity codes in the
sequences. 

6.  A bug in the memory access during the calculation of profiles has been
fixed. (Thanks to Haruna Cofer at SGI).

7. A bug has been fixed in the 'transition weight' option for nucleic acid
sequences. (Thanks to Chanan Rubin at Compugen).

8. An option has been added to read in a series of comparison matrices from a
file. This option is only applicable for protein sequences. For details of the
file format, see the on-line documentation.

9. The MSF output file format has been changed. The sequence weights
calculated by Clustal W are now included in the header.

10. Two bugs in the FAST/APPROXIMATE pairwise alignments have been fixed. One
involved the alignment of new sequences to an existing profile using the fast
pairwise alignment option; the second was caused by changing the default
options for the fast pairwise alignments.

11. A bug in the alignment of a small number of sequences has been fixed.
Previously a Guide Tree was not calculated for less than 4 sequences.


Changes 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 was modified from version 1.7:

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.

                              CLUSTAL REFERENCES
                              ------------------

Details of algorithms, implementation and useful tips on usage of Clustal
programs can be found in the following publications:

Jeanmougin,F., Thompson,J.D., Gouy,M., Higgins,D.G. and Gibson,T.J. (1998)
Multiple sequence alignment with Clustal X. Trends Biochem Sci, 23, 403-5.

Thompson,J.D., Gibson,T.J., Plewniak,F., Jeanmougin,F. and Higgins,D.G. (1997)
The ClustalX windows interface: flexible strategies for multiple sequence 
alignment aided by quality analysis tools. Nucleic Acids Research, 24:4876-4882.

Higgins, D. G., Thompson, J. D. and Gibson, T. J. (1996) Using CLUSTAL for
multiple sequence alignments. Methods Enzymol., 266, 383-402.

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.

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

Higgins,D.G. and Sharp,P.M. (1989) Fast and sensitive multiple sequence
alignments on a microcomputer. CABIOS 5,151-153.

Higgins,D.G. and Sharp,P.M. (1988) CLUSTAL: a package for performing multiple
sequence alignment on a microcomputer. Gene 73,237-244.
