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# clustalw.doc

- DISCLAIMER
- DOCUMENTATION

## DISCLAIMER

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

## DOCUMENTATION

    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 The static arrays used by clustalw for storing the alignment data have been replaced by dynamically allocated memory.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 :-).

### ------------------------------------------------------------------------------