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ClustalW Reference

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.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) 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.doc). 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.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 "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.


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


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.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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