Clustal W version 1.6 March 1996
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@EBI.AC.UK)
EMBL outstation
The European Bioinformatics Institute
Hinxton Hall Hinxton
Cambridge CB10 1RQ
UK
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 (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=3D and
/profile2=3D) 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
- i.e., 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=3Dprotein /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 =3D 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.doc. 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) 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.
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 " " "
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 "-" charcters. 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.
2) 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.doc). WE DO NOT PROVIDE ANY DIRECT MEANS FOR
VIEWING TREES GRAPHICALLY.
3) THE ALIGNMENT ALGORITHMS
The basic algorithm is the same as for Clustal V and is described in
some detail in clustalv.doc. 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 "r 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.
4) 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 =3D -Ln(1 - D - (D*D)/5)
where D is the observed distance and K is the 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)=2E 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 =3D 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 gives values of K for all D between
0.75 and 0.93 in intervals of 0.001 i.e. for D =3D 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 c 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.
5) 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). The basic command line style is very VAX/VMS
like where you put each option after a "/" charcter. Ambiguity and
case handling are primitive.
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 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 and you will see a list with no explanation.
To get (VERY BRIEF) help on command line usage, use the /HELP or
/CHECK options. Otherwise, the command line usage is self explanatory
or is explained in clustalv.doc. 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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