This is the on-line help file for Clustal X (version 2.0 or greater).

It should be named or defined as:
    clustalx.hlp

Toby  Gibson                         EMBL, Heidelberg, Germany.
Des   Higgins                        Conway Institute, UCD, Dublin, Ireland.
Julie Thompson/Francois Jeanmougin   IGBMC, Strasbourg, France.


>>HELP G <<
<H3>
                    General help for CLUSTAL X (2.0)
</H3>

<P>
Clustal X is a windows interface for the ClustalW multiple sequence alignment
program. It provides an integrated environment for performing multiple sequence
and profile alignments and analysing the results. The sequence alignment is
displayed in a window on the screen. A versatile coloring scheme has been
incorporated allowing you to highlight conserved features  in the alignment.
The pull-down menus at the top of the window allow you to select all the
options required for traditional multiple sequence and profile alignment.
</P>

<P>
You can cut-and-paste sequences to change the order of the alignment; you can
select a subset of sequences to be aligned; you can select a sub-range of the
alignment to be realigned and inserted back into the original alignment.
</P>

<P>
Alignment quality analysis can be performed and low-scoring segments or
exceptional residues can be highlighted.
</P>

<P>
ClustalX is available on Linux, Mac and Windows.
</P>



<H4>
SEQUENCE INPUT
</H4>

<P>
Sequences and profiles (a term for pre-existing alignments) are input using 
the FILE menu. Invalid options will be disabled. All sequences must be included
into 1 file. 7 formats are automatically recognised: NBRF/PIR, EMBL/SWISSPROT,
Pearson (Fasta), Clustal (*.aln), GCG/MSF (Pileup), GCG9 RSF and GDE flat file.
All non-alphabetic characters (spaces, digits, punctuation marks) are ignored
except "<CODE>-</CODE>" which is used to indicate a GAP ("<CODE>.</CODE>" in
MSF/RSF).
</P>

<H4>
SEQUENCE / PROFILE ALIGNMENTS
</H4>

<P>
Clustal X has two modes which can be selected using the switch directly above
the sequence display: MULTIPLE ALIGNMENT MODE and PROFILE ALIGNMENT MODE.
</P>

<P>
To do a MULTIPLE ALIGNMENT on a set of sequences, make sure MULTIPLE ALIGNMENT
MODE is selected. A single sequence data area is then displayed. The ALIGNMENT
menu then allows you to either produce a guide tree for the alignment, or to do
a multiple alignment following the guide tree, or to do a full multiple
alignment.
</P>

<P>
In PROFILE ALIGNMENT MODE, two sequence data areas are displayed, allowing
you to align 2 alignments (termed profiles). Profiles are also used to add a
new sequence to an old alignment, or to use secondary structure to guide the
alignment process. GAPS in the old alignments are indicated using the
"<CODE>-</CODE>" character. PROFILES can be input in ANY of the allowed
formats; just use "<CODE>-</CODE>" (or "<CODE>.</CODE>" for MSF/RSF) for each
gap position. In Profile Alignment Mode, a button "Lock Scroll" is displayed
which allows you to scroll the two profiles together using a single scroll
bar. When the Lock Scroll is turned off, the two profiles can be scrolled
independently.
</P>

<H4>
PHYLOGENETIC TREES
</H4>

<P>
Phylogenetic trees can be calculated from old alignments (read in with
"<CODE>-</CODE>" characters to indicate gaps) OR after a multiple alignment
while the alignment is still displayed.
</P>

<H4>
ALIGNMENT DISPLAY
</H4>

<P>
The alignment is displayed on the screen with the sequence names on the left
hand side. The sequence alignment is for display only, it cannot be edited here
(except for changing the sequence order by cutting-and-pasting on the sequence
names). 
</P>

<P>
A ruler is displayed below the sequences, starting at 1 for the first residue
position (residue numbers in the sequence input file are ignored).
</P>

<P>
A line above the alignment is used to mark strongly conserved positions. Three
characters ("<CODE>*</CODE>", "<CODE>:</CODE>" and "<CODE>.</CODE>") are used:
</P>

<P>
"<CODE>*</CODE>" indicates positions which have a single, fully conserved
residue.
</P>

<P>
"<CODE>:</CODE>" indicates that one of the following 'strong' groups is fully
conserved:
</P>

<PRE>
    STA  
    NEQK  
    NHQK  
    NDEQ  
    QHRK  
    MILV  
    MILF  
    HY  
    FYW  
</PRE>

<P>
"<CODE>.</CODE>" indicates that one of the following 'weaker' groups is fully
conserved:
</P>

<PRE>
    CSA  
    ATV  
    SAG  
    STNK  
    STPA  
    SGND  
    SNDEQK  
    NDEQHK  
    NEQHRK  
    FVLIM  
    HFY
</PRE>

<P>
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 =&lt; 0.5 respectively.
</P>

<P>
For profile alignments, secondary structure and gap penalty masks are displayed
above the sequences, if any data is found in the profile input file.
</P>


>>HELP F <<
<H3>
                      Input / Output Files 
</H3>

<P>
LOAD SEQUENCES reads sequences from one of 7 file formats, replacing any
sequences that are already loaded. All sequences must be in 1 file. The formats
that are automatically recognised are: NBRF/PIR, EMBL/SWISSPROT, Pearson
(Fasta), Clustal (*.aln), GCG/MSF (Pileup), GCG9/RSF and GDE flat file.  All
non-alphabetic characters (spaces, digits, punctuation  marks) are ignored
except "<CODE>-</CODE>" which is used to indicate a GAP ("<CODE>.</CODE>" in
MSF/RSF).
</P>

<P>
The program tries to automatically recognise the different file formats used
and to guess whether the sequences are amino acid or nucleotide.  This is not
always foolproof.
</P>

<P>
FASTA and NBRF/PIR formats are recognised by having a "<CODE>></CODE>" as the
first character in the file.  
</P>

<P>
EMBL/Swiss Prot formats are recognised by the letters "<CODE>ID</CODE>" at the
start of the file (the token for the entry name field).
</P>

<P>
CLUSTAL format is recognised by the word <CODE>CLUSTAL</CODE> at the beginning
of the file.
</P>

<P>
GCG/MSF format is recognised by one of the following:
<UL>
<LI>
       the word <CODE>PileUp</CODE> at the start of the file.
</LI>
<LI>
       the word <CODE>!!AA_MULTIPLE_ALIGNMENT</CODE> or
       <CODE>!!NA_MULTIPLE_ALIGNMENT</CODE> at the start of the file.
</LI>
<LI>
       the word <CODE>MSF</CODE> on the first line of the file, and the
       characters <CODE>..</CODE> at the end of this line.
</LI>
</UL>
</P>
 
<P>
GCG/RSF format is recognised by the word <CODE>!!RICH_SEQUENCE</CODE> at the
beginning of the file.
</P>


<P>
If 85% or more of the characters in the sequence are from A,C,G,T,U or N, the
sequence will be assumed to be nucleotide.  This works in 97.3% of cases but
watch out!
</P>

<P>
APPEND SEQUENCES is only valid in MULTIPLE ALIGNMENT MODE. The input sequences
do not replace those already loaded, but are appended at the end of the
alignment.
</P>

<P>
SAVE SEQUENCES AS... offers the user a choice of one of six output formats:
CLUSTAL, NBRF/PIR, GCG/MSF, PHYLIP, NEXUS, GDE or FASTA. All sequences are
written to a single file. Options are available to save a range of the
alignment, switch between UPPER/LOWER case for GDE files, and to output
SEQUENCE NUMBERING for CLUSTAL files. Users can also choose to include the
residue range numbers by appending them to the sequence names.
</P>

<P>
LOAD PROFILE 1 reads sequences in the same 7 file formats, replacing any
sequences already loaded as Profile 1. This option will also remove any
sequences which are loaded in Profile 2.
</P>

<P>
LOAD PROFILE 2 reads sequences in the same 7 file formats, replacing any
sequences already loaded as Profile 2.
</P>

<P>
SAVE PROFILE 1 AS... is similar to the Save Sequences option except that only
those sequences in Profile 1 will be written to the output file.
</P>

<P>
SAVE PROFILE 2 AS... is similar to the Save Sequences option except that only
those sequences in Profile 2 will be written to the output file.
</P>

<P>
WRITE ALIGNMENT AS POSTSCRIPT will write the sequence display to a postscript
format file. This will include any secondary structure / gap penalty mask 
information and the consensus and ruler lines which are displayed on the
screen. The Alignment Quality curve can be optionally included in the output
file.
</P>

<P>
WRITE PROFILE 1 AS POSTSCRIPT is similar to WRITE ALIGNMENT AS POSTSCRIPT
except that only the profile 1 display will be printed.
</P>

<P>
WRITE PROFILE 2 AS POSTSCRIPT is similar to WRITE ALIGNMENT AS POSTSCRIPT
except that only the profile 2 display will be printed.
</P>


<H4>
POSTSCRIPT PARAMETERS
</H4>

<P>
A number of options are available to allow you to configure your postscript
output file.
</P>

<P>
PS COLORS FILE: The exact RGB values required to reproduce the colors used
in the alignment window will vary from printer to printer. A PS colors file
can be specified that contains the RGB values for all the colors required by
each of your postscript printers.
</P>

<P>
By default, Clustal X looks for a file called "<CODE>colprint.par</CODE>" in
the current directory (if your running under UNIX, it then looks in your home
directory, and finally in the directories in your PATH environment
variable). If no PS colors file is found or a color used on the screen is not
defined here, the screen RGB values (from the Color Parameter File) are used.
</P>

<P>
The PS colors file consists of one line for each color to be defined, with the
color name followed by the RGB values (on a scale of 0 to 1). For example,
</P>

<PRE>
    RED          0.9 0.1 0.1
</PRE>

<P>
Blank lines and comments (lines beginning with a "<CODE>#</CODE>" character)
are ignored.
</P>


<P>
PAGE SIZE:  The alignment can be displayed on either A4, A3 or US Letter size
pages.
</P>

<P>
ORIENTATION: The alignment can be displayed on either a landscape or portrait
page.
</P>

<P>
PRINT HEADER: An optional header including the postscript filename, and
creation date can be printed at the top of each page.
</P>

<P>
PRINT QUALITY CURVE: The Alignment Quality curve which is displayed underneath
the alignment on the screen can be included in the postscript output.
</P>

<P>
PRINT RULER: The ruler which is displayed underneath the alignment on the 
screen can be included in the postscript output.
</P>

<P>
PRINT RESIDUE NUMBERS: Sequence residue numbers can be printed at the right
hand side of the alignment.
</P>

<P>
RESIZE TO FIT PAGE: By default, the alignment is scaled to fit the page size
selected. This option can be turned off, in which case a font size of 10 will
be used for the sequences.
</P>

<P>
PRINT FROM POSITION/TO: A range of the alignment can be printed. The default
is to print the full alignment. The first and last residues to be printed are
specified here.
</P>

<P>
USE BLOCK LENGTH: The alignment can be divided into blocks of residues. The
number of residues in a block is specified here. More than one block may then
be printed on a single page. This is useful for long alignments of a small
number of sequences. If the block length is set to 0, The alignment will not
be divided into blocks, but printed across a number of pages.
</P>


>>HELP E <<
<H3>
                          Editing Alignments
</H3>

<P>
Clustal X allows you to change the order of the sequences in the alignment, by
cutting-and-pasting the sequence names.
</P>

<P>
To select a group of sequences to be moved, click on a sequence name and drag
the cursor until all the required sequences are highlighted. Holding down the
Shift key when clicking on the first name will add new sequences to those
already selected.
</P>

<P>
(Options are provided to Select All Sequences, Select Profile 1 or Select 
Profile 2.)
</P>

<P>
The selected sequences can be removed from the alignment by using the EDIT
menu, CUT option.
</P>

<P>
To add the cut sequences back into an alignment, select a sequence by clicking
on the sequence name. The cut sequences will be added to the alignment,
immediately following the selected sequence, by the EDIT menu, PASTE option.
</P>

<P>
To add the cut sequences to an empty alignment (eg. when cutting sequences from
Profile 1 and pasting them to Profile 2), click on the empty sequence name
display area, and select the EDIT menu, PASTE option as before.
</P>

<P>
The sequence selection and sequence range selection can be cleared using the
EDIT menu, CLEAR SEQUENCE SELECTION and CLEAR RANGE SELECTION options
respectively.
</P>

<P>
To search for a string of residues in the sequences, select the sequences to be
searched by clicking on the sequence names. You can then enter the string to
search for by selecting the SEARCH FOR STRING option. If the string is found in
any of the sequences selected, the sequence name and column number is printed
below the sequence display.
</P>

<P>
In PROFILE ALIGNMENT MODE, the two profiles can be merged (normally done after
alignment) by selecting ADD PROFILE 2 TO PROFILE 1. The sequences currently
displayed as Profile 2 will be appended to Profile 1. 
</P>

<P>
The REMOVE ALL GAPS option will remove all gaps from the sequences currently
selected.
WARNING: This option removes ALL gaps, not only those introduced by ClustalX,
but also those that were read from the input alignment file. Any secondary
structure information associated with the alignment will NOT be automatically
realigned.
</P>

<P>
The REMOVE GAP-ONLY COLUMNS will remove those positions in the alignment which
contain gaps in all sequences. This can occur as a result of removing divergent
sequences from an alignment, or if an alignment has been realigned.
</P>


>>HELP M <<
<H3>
                          Multiple Alignments
</H3>

<P>
Make sure MULTIPLE ALIGNMENT MODE is selected, using the switch directly above
the sequence display area. Then, use the ALIGNMENT menu to do multiple
alignments.
</P>

<P>
Multiple alignments are carried out in 3 stages:
</P>
 
<OL>
<LI>
all sequences are compared to each other (pairwise alignments);
</LI>
 
<LI>
a dendrogram (like a phylogenetic tree) is constructed, describing the
approximate groupings of the sequences by similarity (stored in a file).
</LI>
 
<LI>
the final multiple alignment is carried out, using the dendrogram as a guide.
</LI>
</OL>

<P>
The 3 stages are carried out automatically by the DO COMPLETE ALIGNMENT option.
You can skip the first stages (pairwise alignments; guide tree) by using an old
guide tree file (DO ALIGNMENT FROM GUIDE TREE); or you can just produce the
guide tree with no final multiple alignment (PRODUCE GUIDE TREE ONLY).
</P>

<P>
REALIGN SELECTED SEQUENCES is used to realign badly aligned sequences in the
alignment. Sequences can be selected by clicking on the sequence names - see
Editing Alignments for more details. The unselected sequences are then 'fixed'
and a profile is made including only the unselected sequences. Each of the
selected sequences in turn is then realigned to this profile. The realigned
sequences will be displayed as a group at the end the alignment.
</P>

<P>
REALIGN SELECTED SEQUENCE RANGE is used to realign a small region of the 
alignment. A residue range can be selected by clicking on the sequence display
area. A multiple alignment is then performed, following the 3 stages described
above, but only using the selected residue range. Finally the new alignment of
the range is pasted back into the full sequence alignment.
</P>

<P>
By default, gap penalties are used at each end of the subrange in order to 
penalise terminal gaps. If the REALIGN SEGMENT END GAP PENALTIES option is
switched off, gaps can be introduced at the ends of the residue range at no
cost.
</P>

<P>
ALIGNMENT PARAMETERS displays a sub-menu with the following options:
</P>

<P>
RESET NEW GAPS BEFORE ALIGNMENT will remove any new gaps introduced into the
sequences during multiple alignment if you wish to change the parameters and
try again. This only takes effect just before you do a second multiple
alignment. You can make phylogenetic trees after alignment whether or not this
is ON. If you turn this OFF, the new gaps are kept even if you do a second
multiple alignment. This allows you to iterate the alignment gradually.
Sometimes, the alignment is improved by a second or third pass.
</P>

<P>
RESET ALL GAPS BEFORE ALIGNMENT will remove all gaps in the sequences including
gaps which were read in from the sequence input file. This only takes effect
just before you do a second multiple alignment.  You can make phylogenetic
trees after alignment whether or not this is ON.  If you turn this OFF, all
gaps are kept even if you do a second multiple alignment. This allows you to
iterate the alignment gradually.  Sometimes, the alignment is improved by a
second or third pass.
</P>

<P>
PAIRWISE ALIGNMENT PARAMETERS control the speed/sensitivity of the initial
alignments.
</P>

<P>
MULTIPLE ALIGNMENT PARAMETERS control the gaps in the final multiple
alignments.
</P>

<P>
PROTEIN GAP PARAMETERS displays a temporary window which allows you to set
various parameters only used in the alignment of protein sequences.
</P>

<P>
(SECONDARY STRUCTURE PARAMETERS, for use with the Profile Alignment Mode only,
allows you to set various parameters only used with gap penalty masks.)
</P>

<P>
SAVE LOG FILE will write the alignment calculation scores to a file. The log
filename is the same as the input sequence filename, with an extension
"<CODE>.log</CODE>" appended.
</P>

<P>
 ITERATION: A remove first iteration scheme has been added. This can be used to 
 improve the final alignment or improve the alignment at each stage of the progressive
 alignment. During the iteration step each sequence is removed in turn and realigned. If the
 resulting alignment is better than the  previous alignment it is kept. This process is
 repeated until the score converges (the  score is not improved) or until the maximum number of iterations is 
 reached. The user can  iterate at each step of the progressive alignment by setting the 
 iteration parameter to  'Iterate each alignment step' or just on the final alignment 
 by setting the iteration 
 parameter to 'Iterate final alignment'. The default number of iterations is 3.
</P>


<H4>
OUTPUT FORMAT OPTIONS
</H4>

<P>
You can choose from 7 different alignment formats (CLUSTAL, GCG, NBRF/PIR,
PHYLIP, GDE, NEXUS, FASTA).  You can choose more than one (or all 7 if you wish).  
</P>

<P>
CLUSTAL format output is a self explanatory alignment format. It shows the
sequences aligned in blocks. It can be read in again at a later date to (for
example) calculate a phylogenetic tree or add in new sequences by profile
alignment.
</P>

<P>
GCG output can be used by any of the GCG programs that can work on multiple
alignments (e.g. PRETTY, PROFILEMAKE, PLOTALIGN). It is the same as the GCG
.msf format files (multiple sequence file); new in version 7 of GCG.
</P>

<P>
NEXUS format is used by several phylogeny programs, including PAUP and
MacClade.
</P>

<P>
PHYLIP format output can be used for input to the PHYLIP package of Joe 
Felsenstein.  This is a very widely used package for doing every imaginable
form of phylogenetic analysis (MUCH more than the the modest introduction
offered by this program).
</P>

<P>
NBRF/PIR: this is the same as the standard PIR format with ONE ADDITION. Gap
characters "<CODE>-</CODE>" are used to indicate the positions of gaps in the
multiple alignment. These files can be re-used as input in any part of
clustal that allows sequences (or alignments or profiles) to be read in.
</P>

<P>
FASTA: this is included for compatibility with numberous sequence analysis
programs.
</P>

<P>
GDE:  this format is used by the GDE package of Steven Smith and is understood
by SEQLAB in GCG 9 or later.
</P>

<P>
GDE OUTPUT CASE: sequences in GDE format may be written in either upper or
lower case.
</P>
 
<P>
CLUSTALW SEQUENCE NUMBERS: residue numbers may be added to the end of the
alignment lines in clustalw format.
</P>

<P>
OUTPUT ORDER is used to control the order of the sequences in the output
alignments. By default, it uses the order in which the sequences were aligned
(from the guide tree/dendrogram), thus automatically grouping closely related
sequences. It can be switched to be the same as the original input order.
</P>

<P>
PARAMETER OUTPUT: This option will save all your parameter settings in a
parameter file (suffix "<CODE>.par</CODE>") during alignment. The file can be
subsequently used to rerun ClustalW using the same parameters.
</P>

<H3>
ALIGNMENT PARAMETERS
</H3>

<H4>
PAIRWISE ALIGNMENT PARAMETERS
</H4>

<P>
A distance is calculated between every pair of sequences and these are used to
construct the phylogenetic tree which guides the final multiple alignment. The
scores are calculated from separate pairwise alignments. These can be
calculated using 2 methods: dynamic programming (slow but accurate) or by the
method of Wilbur and Lipman (extremely fast but approximate).   
</P>

<P>
You can choose between the 2 alignment methods using the PAIRWISE ALIGNMENTS
option. The slow/accurate method is fast enough for short sequences but will be
VERY SLOW for many (e.g. >100) long (e.g. >1000 residue) sequences.   
</P>


<H4>
SLOW-ACCURATE alignment parameters:
</H4>

<P>
These parameters do not have any affect on the speed of the alignments. They
are used to give initial alignments which are then rescored to give percent
identity scores. These % scores are the ones which are displayed on the 
screen. The scores are converted to distances for the trees.
</P>

<P>
Gap Open Penalty: the penalty for opening a gap in the alignment.
</P>

<P>
Gap Extension Penalty: the penalty for extending a gap by 1 residue.
</P>

<P>
Protein Weight Matrix: the scoring table which describes the similarity of 
each amino acid to each other.
</P>

<P>
Load protein matrix: allows you to read in a comparison table from a file.
</P>

<P>
DNA weight matrix: the scores assigned to matches and mismatches (including
IUB ambiguity codes).
</P>

<P>
Load DNA matrix: allows you to read in a comparison table from a file.
</P>

<P>
See the Multiple alignment parameters, MATRIX option below for details of the
matrix input format.
</P>


<H4>
FAST-APPROXIMATE alignment parameters:
</H4>

<P>
These similarity scores are calculated from fast, approximate, global align-
ments, which are controlled by 4 parameters. 2 techniques are used to make
these alignments very fast: 1) only exactly matching fragments (k-tuples) are
considered; 2) only the 'best' diagonals (the ones with most k-tuple matches)
are used.
</P>

<P>
GAP PENALTY:   This is a penalty for each gap in the fast alignments. It has
little effect on the speed or sensitivity except for extreme values.
</P>

<P>
K-TUPLE SIZE:  This is the size of exactly matching fragment that is used. 
INCREASE for speed (max= 2 for proteins; 4 for DNA), DECREASE for sensitivity.
For longer sequences (e.g. >1000 residues) you may wish to increase the
default.
</P>

<P>
TOP DIAGONALS: The number of k-tuple matches on each diagonal (in an imaginary
dot-matrix plot) is calculated. Only the best ones (with most matches) are used
in the alignment. This parameter specifies how many. Decrease for speed;
increase for sensitivity.
</P>

<P>
WINDOW SIZE:  This is the number of diagonals around each of the 'best' 
diagonals that will be used. Decrease for speed; increase for sensitivity.
</P>


<H4>
MULTIPLE ALIGNMENT PARAMETERS
</H4>

<P>
These parameters control the final multiple alignment. This is the core of the
program and the details are complicated. To fully understand the use of the
parameters and the scoring system, you will have to refer to the documentation.
</P>

<P>
Each step in the final multiple alignment consists of aligning two alignments 
or sequences. This is done progressively, following the branching order in the
GUIDE TREE. The basic parameters to control this are two gap penalties and the
scores for various identical/non-indentical residues. 
</P>

<P>
The GAP OPENING and EXTENSION PENALTIES can be set here. These control the 
cost of opening up every new gap and the cost of every item in a gap.  
Increasing the gap opening penalty will make gaps less frequent. Increasing 
the gap extension penalty will make gaps shorter. Terminal gaps are not 
penalised.
</P>

<P>
The DELAY DIVERGENT SEQUENCES switch delays the alignment of the most distantly
related sequences until after the most closely related sequences have  been
aligned. The setting shows the percent identity level required to delay the
addition of a sequence; sequences that are less identical than this level to
any other sequences will be aligned later.
</P>

<P>
The TRANSITION WEIGHT gives transitions (A&lt;-->G or C&lt;-->T
i.e. purine-purine or pyrimidine-pyrimidine substitutions) a weight between 0
and 1; a weight of zero means that the transitions are scored as mismatches,
while a weight of 1 gives the transitions the 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. The default is set to
0.5.
</P>

<P>
The PROTEIN WEIGHT MATRIX option allows you to choose a series of weight
matrices. For protein alignments, you use a weight matrix to determine the
similarity of non-identical amino acids. For example, Tyr aligned with Phe is
usually judged to be 'better' than Tyr aligned with Pro.
</P>

<P>
There are three 'in-built' series of weight matrices offered. Each consists of
several matrices which work differently at different evolutionary distances. To
see the exact details, read the documentation. Crudely, we store several
matrices in memory, spanning the full range of amino acid distance (from almost
identical sequences to highly divergent ones). For very similar sequences, it
is best to use a strict weight matrix which only gives a high score to
identities and the most favoured conservative substitutions. For more divergent
sequences, it is appropriate to use 'softer' matrices which give a high score
to many other frequent substitutions.
</P>

<OL>
<LI>
BLOSUM (Henikoff). These matrices appear to be the best available for 
carrying out data base similarity (homology searches). The matrices currently
used are: Blosum 80, 62, 45 and 30. BLOSUM was the default in earlier Clustal X
versions.
</LI>

<LI>
PAM (Dayhoff). These have been extremely widely used since the late '70s. We
currently use the PAM 20, 60, 120, 350 matrices.
</LI>

<LI>
GONNET. These matrices were derived using almost the same procedure as the
Dayhoff one (above) but are much more up to date and are based on a far larger
data set. They appear to be more sensitive than the Dayhoff series. We
currently use the GONNET 80, 120, 160, 250 and 350 matrices. This series is the
default for Clustal X version 1.8.
</LI>
</OL>

<P>
We also supply an identity matrix which gives a score of 10 to two identical 
amino acids and a score of zero otherwise. This matrix is not very useful.
</P>

<P>
Load protein matrix: allows you to read in a comparison matrix from a file.
This can be either a single matrix or a series of matrices (see below for
format). 
</P>


<P>
DNA WEIGHT MATRIX option allows you to select a single matrix (not a series)
used for aligning nucleic acid sequences. Two hard-coded matrices are
available:
</P>

<OL>
<LI>
IUB. This is the default scoring matrix used by BESTFIT 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.
</LI>

<LI>
CLUSTALW(1.6). A previous system used by ClustalW, in which matches score
1.0 and mismatches score 0. All matches for IUB symbols also score 0.
</LI>
</OL>

<P>
Load DNA matrix: allows you to read in a nucleic acid comparison matrix from a
file (just one matrix, not a series).
</P>


<P>
SINGLE MATRIX INPUT FORMAT
The format used for a single matrix is the same as the BLAST program. The
scores in the new weight matrix should be similarities. You can use negative as
well as positive values if you wish, although the matrix will be automatically
adjusted to all positive scores, unless the NEGATIVE MATRIX option is selected.
Any lines beginning with a "<CODE>#</CODE>" character are assumed to be
comments. The first non-comment line should contain a list of amino acids in
any order, using the 1 letter code, followed by a "<CODE>*</CODE>"
character. This should be followed by a square matrix of scores, with one row
and one column for each amino acid. The last row and column of the matrix
(corresponding to the "<CODE>*</CODE>" character) contain the minimum score
over the whole matrix.
</P>

<P>
MATRIX SERIES INPUT FORMAT
ClustalX uses different matrices depending on the mean percent identity of the
sequences to be aligned. You can specify a series of matrices and the range of
the percent identity for each matrix in a matrix series file. The file is
automatically recognised by the word CLUSTAL_SERIES at the beginning of the
file. Each matrix in the series is then specified on one line which should
start with the word MATRIX. This is followed by the lower and upper limits of
the sequence percent identities for which you want to apply the matrix. The
final entry on the matrix line is the filename of a Blast format matrix file
(see above for details of the single matrix file format).
</P>

<P>
Example.
</P>

<PRE>
    CLUSTAL_SERIES
     
    MATRIX 81 100 /us1/user/julie/matrices/blosum80
    MATRIX 61 80 /us1/user/julie/matrices/blosum62
    MATRIX 31 60 /us1/user/julie/matrices/blosum45
    MATRIX 0 30 /us1/user/julie/matrices/blosum30
</PRE>

<H4>
PROTEIN GAP PARAMETERS
</H4>

<P>
RESIDUE SPECIFIC PENALTIES are amino acid specific gap penalties that reduce or
increase the gap opening penalties at each position in the alignment or 
sequence. See the documentation for details. As an example, positions that are
rich in glycine are more likely to have an adjacent gap than positions that are
rich in valine.
</P>

<P>
HYDROPHILIC GAP PENALTIES are used to increase the chances of a gap within a
run (5 or more residues) of hydrophilic amino acids; these are likely to be
loop or random coil regions where gaps are more common. The residues that are
'considered' to be hydrophilic can be entered in HYDROPHILIC RESIDUES.
</P>

<P>
GAP SEPARATION DISTANCE tries to decrease the chances of gaps being too close
to each other. Gaps that are less than this distance apart are penalised more
than other gaps. This does not prevent close gaps; it makes them less frequent,
promoting a block-like appearance of the alignment.
</P>

<P>
END GAP SEPARATION treats end gaps just like internal gaps for the purposes of
avoiding gaps that are too close (set by GAP SEPARATION DISTANCE above). If you
turn this off, end gaps will be ignored for this purpose. This is useful when
you wish to align fragments where the end gaps are not biologically meaningful.
</P>


>>HELP P <<
<H3>
                   Profile and Structure Alignments
</H3>
   
<P>
By PROFILE ALIGNMENT, we mean alignment using existing alignments. Profile 
alignments allow you to store alignments of your favourite sequences and add
new sequences to them in small bunches at a time. A profile is simply an
alignment of one or more sequences (e.g. an alignment output file from Clustal
X). Each input can be a single sequence. One or both sets of input sequences
may include secondary structure assignments or gap penalty masks to guide the
alignment. 
</P>

<P>
Make sure PROFILE ALIGNMENT MODE is selected, using the switch directly above
the sequence display area. Then, use the ALIGNMENT menu to do profile and
secondary structure alignments.
</P>

<P>
The profiles can be in any of the allowed input formats with "<CODE>-</CODE>"
characters used to specify gaps (except for GCG/MSF where "<CODE>.</CODE>" is
used).
</P>

<P>
You have to load the 2 profiles by choosing FILE, LOAD PROFILE 1 and  LOAD
PROFILE 2. Then ALIGNMENT, ALIGN PROFILE 2 TO PROFILE 1 will align the 2
profiles to each other. Secondary structure masks in either profile can be used
to guide the alignment. This option compares all the sequences in profile 1
with all the sequences in profile 2 in order to build guide trees which will be
used to calculate sequence weights, and select appropriate alignment parameters
for the final profile alignment.
</P>

<P>
You can skip the first stage (pairwise alignments; guide trees) by using old
guide tree files (ALIGN PROFILES FROM GUIDE TREES). 
</P>

<P>
The ALIGN SEQUENCES TO PROFILE 1 option will take the sequences in the second
profile and align them to the first profile, 1 at a time.  This is useful to
add some new sequences to an existing alignment, or to align a set of sequences
to a known structure. In this case, the second profile set need not be
pre-aligned.
</P>

<P>
You can skip the first stage (pairwise alignments; guide tree) by using an old
guide tree file (ALIGN SEQUENCES TO PROFILE 1 FROM TREE). 
</P>

<P>
SAVE LOG FILE will write the alignment calculation scores to a file. The log
filename is the same as the input sequence filename, with an extension
"<CODE>.log</CODE>" appended.
</P>

<P>
The alignment parameters can be set using the ALIGNMENT PARAMETERS menu,
Pairwise Parameters, Multiple Parameters and Protein Gap Parameters options.
These are EXACTLY the same parameters as used by the general, automatic
multiple alignment procedure. The general multiple alignment procedure is
simply a series of profile alignments. Carrying out a series of profile
alignments on larger and larger groups of sequences, allows you to manually
build up a complete alignment, if necessary editing intermediate alignments.
</P>

<H4>
SECONDARY STRUCTURE PARAMETERS
</H4>

<P>
Use this menu to set secondary structure options. If a solved structure is
known, it can be used to guide the alignment by raising gap penalties within
secondary structure elements, so that gaps will preferentially be inserted into
unstructured surface loop regions. Alternatively, a user-specified gap penalty
mask can be supplied for a similar purpose.
</P>

<P>
A gap penalty mask is a series of numbers between 1 and 9, one per position in 
the alignment. Each number specifies how much the gap opening penalty is to be 
raised at that position (raised by multiplying the basic gap opening penalty
by the number) i.e. a mask figure of 1 at a position means no change
in gap opening penalty; a figure of 4 means that the gap opening penalty is
four times greater at that position, making gaps 4 times harder to open.
</P>

<P>
The format for gap penalty masks and secondary structure masks is explained in
a separate help section.
</P>


>>HELP B << 
<H3>
            Secondary Structure / Gap Penalty Masks
</H3>

<P>
The use of secondary structure-based penalties has been shown to improve  the
accuracy of sequence alignment. Clustal X now allows secondary structure/gap
penalty masks to be supplied with the input sequences used during profile
alignment. (NB. The secondary structure information is NOT used during multiple
sequence alignment). The masks work by raising gap penalties in specified
regions (typically secondary structure elements) so that gaps are
preferentially opened in the less well conserved regions (typically surface
loops).
</P>

<P>
The USE PROFILE 1(2) SECONDARY STRUCTURE / GAP PENALTY MASK options control
whether the input 2D-structure information or gap penalty masks will be used
during the profile alignment.
</P>

<P>
The OUTPUT options control whether the secondary structure and gap penalty
masks should be included in the Clustal X output alignments. Showing both is
useful for understanding how the masks work. The 2D-structure information is
itself useful in judging the alignment quality and in seeing how residue
conservation patterns vary with secondary structure. 
</P>

<P>
The HELIX and STRAND GAP PENALTY options provide the value for raising the gap
penalty at core Alpha Helical (A) and Beta Strand (B) residues. In CLUSTAL
format, capital residues denote the A and B core structure notation. Basic gap
penalties are multiplied by the amount specified.
</P>

<P>
The LOOP GAP PENALTY option provides the value for the gap penalty in Loops.
By default this penalty is not raised. In CLUSTAL format, loops are specified
by "<CODE>.</CODE>" in the secondary structure notation.
</P>

<P>
The SECONDARY STRUCTURE TERMINAL PENALTY provides the value for setting the gap
penalty at the ends of secondary structures. Ends of secondary structures are
known to grow or shrink, comparing related structures. Therefore by default
these are given intermediate values, lower than the core penalties. All
secondary structure read in as lower case in CLUSTAL format gets the reduced
terminal penalty.
</P>

<P>
The HELIX and STRAND TERMINAL POSITIONS options specify the range of structure
termini for the intermediate penalties. In the alignment output, these are
indicated as lower case. For Alpha Helices, by default, the range spans the 
end-helical turn (3 residues). For Beta Strands, the default range spans the
end residue and the adjacent loop residue, since sequence conservation often
extends beyond the actual H-bonded Beta Strand.
</P>

<P>
Clustal X can read the masks from SWISS-PROT, CLUSTAL or GDE format input
files. For many 3-D protein structures, secondary structure information is
recorded in the feature tables of SWISS-PROT database entries. You should
always check that the assignments are correct - some are quite inaccurate.
Clustal X looks for SWISS-PROT HELIX and STRAND assignments e.g.
</P>

<PRE>
    FT   HELIX       100    115
    FT   STRAND      118    119
</PRE>

<P>
The structure and penalty masks can also be read from CLUSTAL alignment format 
as comment lines beginning "<CODE>!SS_</CODE>" or "<CODE>!GM_</CODE>" e.g.
</P>

<PRE>
    !SS_HBA_HUMA    ..aaaAAAAAAAAAAaaa.aaaAAAAAAAAAAaaaaaaAaaa.........aaaAAAAAA
    !GM_HBA_HUMA    112224444444444222122244444444442222224222111111111222444444
    HBA_HUMA        VLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLSHGSAQVKGHGK
</PRE>

<P>
Note that the mask itself is a set of numbers between 1 and 9 each of which is 
assigned to the residue(s) in the same column below. 
</P>

<P>
In GDE flat file format, the masks are specified as text and the names must
begin with "<CODE>SS_</CODE>" or "<CODE>GM_</CODE>".
</P>

<P>
Either a structure or penalty mask or both may be used. If both are included
in an alignment, the user will be asked which is to be used.
</P>


>>HELP T <<
<H3>
                            Phylogenetic Trees
</H3>

<P>
Before calculating a tree, you must have an ALIGNMENT in memory. This can be
input using the FILE menu, LOAD SEQUENCES option or you should have just
carried out a full multiple alignment and the alignment is still in memory.
Remember YOU MUST ALIGN THE SEQUENCES FIRST!!!!
</P>

<P>
 The UPGMA algorithm has been added to allow faster tree construction. The user now
 has the choice of using Neighbour Joining or UPGMA. The default is still NJ, but the
 user can change this by setting the clustering parameter.
</P>

<P>
To calculate a tree, use the DRAW TREE option. This gives an UNROOTED tree
and all branch lengths when using the NJ method. The root of the tree can only be inferred by using an
outgroup (a sequence that you are certain branches at the outside of the
tree... certain on biological grounds) OR if you assume a degree of constancy
in the 'molecular clock', you can place the root in the 'middle' of the tree 
(roughly equidistant from all tips). The UPGMA algorithm generates a rooted tree.
</P>

<P>
BOOTSTRAP N-J TREE uses a method for deriving confidence values for the 
groupings in a tree (first adapted for trees by Joe Felsenstein). It involves
making N random samples of sites from the alignment (N should be LARGE, e.g.
500 - 1000); drawing N trees (1 from each sample) and counting how many times
each grouping from the original tree occurs in the sample trees. You can set N
using the NUMBER OF BOOTSTRAP TRIALS option in the BOOTSTRAP TREE window. In
practice, you should use a large number of bootstrap replicates (1000 is
recommended, even if it means running the program for an hour on a slow 
computer). You can also supply a seed number for the random number generator
here. Different runs with the same seed will give the same answer. See the
documentation for more details.
</P>

<P>
EXCLUDE POSITIONS WITH GAPS? With this option, any alignment positions where
ANY of the sequences have a gap will be ignored. This means that 'like' will
be compared to 'like' in all distances, which is highly desirable. It also
automatically throws away the most ambiguous parts of the alignment, which are
concentrated around gaps (usually). The disadvantage is that you may throw away
much of the data if there are many gaps (which is why it is difficult for us to
make it the default).  
</P>

<P>
CORRECT FOR MULTIPLE SUBSTITUTIONS? For small divergence (say &lt;10%) this
option makes no difference. For greater divergence, this option corrects for
the fact that observed distances underestimate actual evolutionary
distances. This is because, as sequences diverge, more than one substitution
will happen at many sites. However, you only see one difference when you look
at the present day sequences. Therefore, this option has the effect of
stretching branch lengths in trees (especially long branches). The corrections
used here (for DNA or proteins) are both due to Motoo Kimura. See the
documentation for details.
</P>

<P>
Where possible, this option should be used. However, for VERY divergent
sequences, the distances cannot be reliably corrected. You will be warned if
this happens. Even if none of the distances in a data set exceed the reliable
threshold, if you bootstrap the data, some of the bootstrap distances may
randomly exceed the safe limit.  
</P>

<P>
SAVE LOG FILE will write the tree calculation scores to a file. The log
filename is the same as the input sequence filename, with an extension
"<CODE>.log</CODE>" appended.
</P>

<H4>
OUTPUT FORMAT OPTIONS
</H4>

<P>
Four different formats are allowed. None of these displays the tree visually.
You can display the tree using the NJPLOT program distributed with Clustal X
OR get the PHYLIP package and use the tree drawing facilities there. 
</P>
 
<OL>
<LI>
CLUSTAL FORMAT TREE. This format is verbose and lists all of the distances
between the sequences and the number of alignment positions used for each. The
tree is described at the end of the file. It lists the sequences that are
joined at each alignment step and the branch lengths. After two sequences are
joined, it is referred to later as a NODE. The number of a NODE is the number
of the lowest sequence in that NODE.   
</LI>

<LI>
PHYLIP FORMAT TREE. This format is the New Hampshire format, used by many
phylogenetic analysis packages. It consists of a series of nested parentheses,
describing the branching order, with the sequence names and branch lengths. It
can be read by the NJPLOT program distributed with ClustalX. It can also be
used by the RETREE, DRAWGRAM and DRAWTREE programs of the PHYLIP package to see
the trees graphically. This is the same format used during multiple alignment
for the guide trees. Some other packages that can read and display New
Hampshire format are TreeTool, TreeView, and Phylowin.
</LI>

<LI>
PHYLIP DISTANCE MATRIX. This format just outputs a matrix of all the
pairwise distances in a format that can be used by the PHYLIP package. It used
to be useful when one could not produce distances from protein sequences in the
Phylip package but is now redundant (PROTDIST of Phylip 3.5 now does this).
</LI>

<LI>
NEXUS FORMAT TREE. This format is used by several popular phylogeny
programs, including PAUP and MacClade. The format is described fully in:
Maddison, D. R., D. L. Swofford and W. P. Maddison.  1997.
NEXUS: an extensible file format for systematic information.
Systematic Biology 46:590-621.
</LI>
</OL>

<P>
BOOTSTRAP LABELS ON: By default, the bootstrap values are correctly placed on
the tree branches of the phylip format output tree. The toggle allows them to
be placed on the nodes, which is incorrect, but some display packages (e.g.
TreeTool, TreeView and Phylowin) only support node labelling but not branch
labelling. Care should be taken to note which branches and labels go together. 
</P>


>>HELP C <<
<H3>
                               Colors
</H3>

<P>
Clustal X provides a versatile coloring scheme for the sequence alignment 
display. The sequences (or profiles) are colored automatically, when they are
loaded. Sequences can be colored either by assigning a color to specific
residues, or on the basis of an alignment consensus. In the latter case, the
alignment consensus is calculated automatically, and the residues in each
column are colored according to the consensus character assigned to that
column. In this way, you can choose to highlight, for example, conserved
hydrophylic or hydrophobic positions in the alignment.
</P>

<P>
The 'rules' used to color the alignment are specified in a COLOR
PARAMETER FILE. Clustal X automatically looks for a file called
"<CODE>colprot.xml</CODE>" for protein sequences or
"<CODE>coldna.xml</CODE>" for DNA, in the installation directory and
then. (Under UNIX, it then looks in your home directory). Under Mac OS
X you can locate these files by clicking on the ClustalX application
while holding down the Ctrl key and selecting "Show package
contents". The files are located in the "Contents/MacOS" folder.
</P>

<P>
By default, if no color parameter file is found, protein sequences are colored
by residue as follows:
</P>

<PRE>
    Color                   Residue Code

    ORANGE                  GPST
    RED                     HKR
    BLUE                    FWY
    GREEN                   ILMV
</PRE>

In the case of DNA sequences, the default colors are as follows:

<PRE>
    Color                   Residue Code

    ORANGE                  A
    RED                     C
    BLUE                    T
    GREEN                   G
</PRE>


<P>
The default BACKGROUND COLORING option shows the sequence residues using a
black character on a colored background. It can be switched off to show
residues as a colored character on a white background. 
</P>

<P>
Either BLACK AND WHITE or DEFAULT COLOR options can be selected. The Color
option looks first for the color parameter file (as described above) and, if no
file is found, uses the default residue-specific colors.
</P>

<P>
You can specify your own coloring scheme by using the LOAD COLOR PARAMETER FILE
option. The format of the color parameter file is described below.
</P>

<H4>
COLOR PARAMETER FILE
</H4>

<P>
This file is an xml file divided into 3 sections:
</P>

<OL>
<LI>
the names and RGB values of the colors (rgbindex)
</LI>

<LI>
the rules for calculating the consensus (consensus)
</LI>

<LI>
the rules for assigning colors to the residues (colorrules)
</LI>
</OL>
 

<P>
An example file is given here.
</P>

<PRE>
&lt;?xml version="1.0" encoding="UTF-8"?&gt;
&lt;colorparam&gt;
   &lt;rgbindex&gt;
      &lt;color name = "RED" red = "229" green = "51" blue = "25"&gt;&lt;/color&gt;
      ...
      &lt;color name = "ORANGE" red = "229" green = "153" blue = "76"&gt;&lt;/color&gt;
   &lt;/rgbindex&gt;
   &lt;consensus&gt;
      &lt;condition name = "%" cutoffpercent = "60" residues = "wlvimafcyhp"&gt;&lt;/condition&gt;
      ...
       &lt;condition name = "Y" cutoffpercent = "85" residues = "y"&gt;&lt;/condition&gt;
   &lt;/consensus&gt;
   &lt;colorrules&gt;
      &lt;resrule residue = "g" colorname = "ORANGE" conditions = ""&gt;&lt;/resrule&gt;
      ...
      &lt;resrule residue = "r" colorname = "RED" conditions = "+KRQ"&gt;&lt;/resrule&gt;
    &lt;/colorrules&gt;
&lt;/colorparam&gt;
</PRE>

<P>
The RGB index section is optional (identified by
<CODE>&lt;rgbindex&gt;</CODE>). If this section exists, each color used in the
file must be named and the RGB values specified (on a scale from 0 to
255). If the RGB index section is not found, the following set of
hard-coded colors will be used.
</P>

<PRE>
  RED     229  51  25
  BLUE     25 127 229
  GREEN    25 204  25
  CYAN     25 178 178
  PINK    229 127 127
  MAGENTA 204  76 204
  YELLOW  204 204   0
  ORANGE  229 153  76
</PRE>

<P>
The consensus section is optional and is identified by the header
<CODE>&lt;consensus&gt;</CODE>. It defines how the consensus is calculated.
</P>
 
<P>
The format of each consensus parameter is:
</P>
 
<PRE>
   &lt;condition name = "C" cutoffpercent = "N" residues = "RESIDUE_LIST"&gt;&lt;/condition&gt;
   where
       C             is a character used to identify the parameter.
       N             is an integer value used as the percentage cutoff point.
       RESIDUE_LIST  is a list of residues.
</PRE>
 

<P>
For example:

   <CODE>&lt;condition name = "#" cutoffpercent = "80" residues = "wlvimafcyhp"&gt;&lt;/condition&gt;</CODE>
</P>

<P>
will assign a consensus character "<CODE>#</CODE>" to any column in the
alignment which contains more than 80% of the residues w,l,v,i,m,a,f,c,y,h and p.
</P>
        
<P>
The third section is identified by the header <CODE>&lt;colorrules&gt;</CODE>, and defines
how colors are assigned to each residue in the alignment.
</P>

<P>
The color rules section has the following format:
</P>

<PRE>
   &lt;resrule residue = "R" colorname = "COLOR" conditions = "RESIDUE_LIST"&gt;&lt;/resrule&gt;
   where
      R             is a character used to denote a residue.
      COLOR         is one of the above defined colors.
      RESIDUE_LIST  is a list of residues
</PRE>
 

<P>
Examples:
</P>

<P>
    <CODE>&lt;resrule residue = "g" colorname = "ORANGE" conditions = ""&gt;&lt;/resrule&gt;</CODE>
</P>

<P>
will color all glycines ORANGE, regardless of the consensus.
</P>

<P>
     <CODE>&lt;resrule residue = "k" colorname = "RED" conditions = "+KRQ"&gt;&lt;/resrule&gt;</CODE>
</P>

<P>
will color BLUE any Lysine which is found in a column with a consensus of
-, R or Q.
</P>
 

>>HELP Q <<
<H3>
                       Alignment Quality Analysis
</H3>

<H4>
QUALITY SCORES
</H4>

<P>
Clustal X provides an indication of the quality of an alignment by plotting
a 'conservation score' for each column of the alignment. A high score indicates
a well-conserved column; a low score indicates low conservation. The quality
curve is drawn below the alignment.
</P>

<P>
Two methods are also provided to indicate single residues or sequence segments
which score badly in the alignment.
</P>
 
<P>
Low-scoring residues are expected to occur at a moderate frequency in all the
sequences because of their steady divergence due to the natural processes of
evolution. The most divergent sequences are likely to have the most outliers.
However, the highlighted residues are especially useful in pointing to
sequence misalignments. Note that clustering of highlighted residues is a
strong indication of misalignment. This can arise due to various reasons, for
example:
</P>
 

<OL>
<LI>
        Partial or total misalignments caused by a failure in the
        alignment algorithm. Usually only in difficult alignment cases.
</LI>
 

<LI>
        Partial or total misalignments because at least one of the
        sequences in the given set is partly or completely unrelated to the
        other sequences. It is up to the user to check that the set of
        sequences are alignable.
</LI>

<LI>
        Frameshift translation errors in a protein sequence causing local
        mismatched regions to be heavily highlighted. These are surprisingly
        common in database entries. If suspected, a 3-frame translation of
        the source DNA needs to be examined.
</LI>
</OL>
 
<P>
Occasionally, highlighted residues may point to regions of some biological
significance. This might happen for example if a protein alignment contains a
sequence which has acquired new functions relative to the main sequence set. It
is important to exclude other explanations, such as error or the natural
divergence of sequences, before invoking a biological explanation.
</P>

<H4>
LOW-SCORING SEGMENTS
</H4>

<P>
Unreliable regions in the alignment can be highlighted using the Low-Scoring
Segments option. A sequence-weighted profile is used to indicate any segments
in the sequences which score badly. Because the profile calculation may take
some time, an option is provided to calculate LOW-SCORING SEGMENTS. The 
segment display can then be toggled on or off without having to repeat the
time-consuming calculations.
</P>

<P>
For details of the low-scoring segment calculation, see the CALCULATION section
below.
</P>


<H4>
LOW-SCORING SEGMENT PARAMETERS
</H4>

<P>
MINIMUM LENGTH OF SEGMENTS: short segments (or even single residues) can be
hidden by increasing the minimum length of segments which will be displayed.
</P>

<P>
DNA MARKING SCALE is used to remove less significant segments from the 
highlighted display. Increase the scale to display more segments; decrease the
scale to remove the least significant.
</P>


<P>
PROTEIN WEIGHT MATRIX: the scoring table which describes the similarity of each
amino acid to each other. The matrix is used to calculate the sequence-
weighted profile scores. There are four 'in-built' Log-Odds matrices offered:
the Gonnet PAM 80, 120, 250, 350 matrices. A more stringent matrix which only
gives a high score to identities and the most favoured conservative
substitutions, may be more suitable when the sequences are closely related. For
more divergent sequences, it is appropriate to use 'softer' matrices which give
a high score to many other frequent substitutions. This  option automatically
recalculates the low-scoring segments.
</P>


<P>
DNA WEIGHT MATRIX: Two hard-coded matrices are available:
</P>

<OL>
<LI>
IUB. This is the default scoring matrix used by BESTFIT for the comparison
of nucleic acid sequences. X's and N's are treated as matches to any IUB
ambiguity symbol. All matches score 1.0; all mismatches for IUB symbols score
0.9.
</LI>

<LI>
CLUSTALW(1.6). The previous system used by ClustalW, in which matches score
1.0 and mismatches score 0. All matches for IUB symbols also score 0. 
</LI>

<LI>
A new matrix can be read from a file on disk, if the filename consists only
of lower case characters. The values in the new weight matrix should be
similarities and should be NEGATIVE for infrequent substitutions.
</LI>
</OL>
 
<P>
INPUT FORMAT. The format used for a new matrix is the same as the BLAST
program. Any lines beginning with a "<CODE>#</CODE>" character are assumed to
be comments. The first non-comment line should contain a list of amino acids
in any order, using the 1 letter code, followed by a "<CODE>*</CODE>"
character. This should be followed by a square matrix of scores, with one row
and one column for each amino acid. The last row and column of the matrix
(corresponding to the "<CODE>*</CODE>" character) contain the minimum score
over the whole matrix.
</P>

<H4>
QUALITY SCORE PARAMETERS
</H4>

<P>
You can customise the column 'quality scores' plotted underneath the alignment
display using the following options.
</P>

<P>
SCORE PLOT SCALE: this is a scalar value from 1 to 10, which can be used to
change the scale of the quality score plot. 
</P>

<P>
RESIDUE EXCEPTION CUTOFF: this is a scalar value from 1 to 10, which can be
used to change the number of residue exceptions which are highlighted in the
alignment display. (For an explanation of this cutoff, see the CALCULATION OF
RESIDUE EXCEPTIONS section below.)
</P>

<P>
PROTEIN WEIGHT MATRIX: the scoring table which describes the similarity of 
each amino acid to each other. 
</P>
 
<P>
DNA WEIGHT MATRIX: two hard-coded matrices are available: IUB and
CLUSTALW(1.6).
</P>

<P>
For more information about the weight matrices, see the help above for
the Low-scoring Segments Weight Matrix.
</P>

<P>
For details of the quality score calculations, see the CALCULATION section
below.
</P>


<STRONG>
SHOW LOW-SCORING SEGMENTS
</STRONG>
                       
<P>
The low-scoring segment display can be toggled on or off. This option does not
recalculate the profile scores.
</P>


<STRONG>
SHOW EXCEPTIONAL RESIDUES
</STRONG>
                       
<P>
This option highlights individual residues which score badly in the alignment
quality calculations. Residues which score exceptionally low are highlighted by
using a white character on a grey background.
</P>

<STRONG>
SAVE QUALITY SCORES TO FILE
</STRONG>

<P>
The quality scores that are plotted underneath the alignment display can also
be saved in a text file. Each column in the alignment is written on one line in
the output file, with the value of the quality score at the end of the line.
Only the sequences currently selected in the display are written to the file.
One use for quality scores is to color residues in a protein structure by
sequence conservation. In this way conserved surface residues can be
highlighted to locate functional regions such as ligand-binding sites.
</P>


<H4>
CALCULATION OF QUALITY SCORES
</H4>

<P>
Suppose we have an alignment of m sequences of length n. Then, the alignment
can be written as:
</P>

<PRE>
    A11 A12 A13 .......... A1n
    A21 A22 A23 .......... A2n
    .
    .
    Am1 Am2 Am3 .......... Amn
</PRE>

<P>
We also have a residue comparison matrix of size R where C(i,j) is the score
for aligning residue i with residue j.
</P>

<P>
We want to calculate a score for the conservation of the jth position in the
alignment.
</P>

<P>
To do this, we define an R-dimensional sequence space. For the jth position in 
the alignment, each sequence consists of a single residue which is assigned a
point S in the space. S has R dimensions, and for sequence i, the rth dimension
is defined as:
</P>

<PRE>
    Sr =    C(r,Aij)
</PRE>

<P>
We then calculate a consensus value for the jth position in the alignment. This
value X also has R dimensions, and the rth dimension is defined as:
</P>

<PRE>
    Xr = (   SUM   (Fij * C(i,r)) ) / m
           1&lt;=i&lt;=R
</PRE>

<P>
where Fij is the count of residues i at position j in the alignment.
</P>

<P>
Now we can calculate the distance Di between each sequence i and the consensus 
position X in the R-dimensional space.
</P>

<PRE>
    Di = SQRT   (   SUM   (Xr - Sr)(Xr - Sr) )
                  1&lt;=i&lt;=R
</PRE>

<P>
The quality score for the jth position in the alignment is defined as the mean
of the sequence distances Di.
</P>

<P>
The score is normalised by multiplying by the percentage of sequences which
have residues (and not gaps) at this position.
</P>

<H4>
CALCULATION OF RESIDUE EXCEPTIONS
</H4>

<P>
The jth residue of the ith sequence is considered as an exception if the
distance Di of the sequence from the consensus value P is greater than (Upper
Quartile + Inter Quartile Range * Cutoff). The value used as a cutoff for
displaying exceptions can be set from the SCORE PARAMETERS menu. A high cutoff
value will only display very significant exceptions; a low value will allow
more, less significant, exceptions to be highlighted.
</P>

<P>
(NB. Sequences which contain gaps at this position are not included in the
exception calculation.)
</P>


<H4>
CALCULATION OF LOW-SCORING SEGMENTS
</H4>

<P>
Suppose we have an alignment of m sequences of length n. Then, the alignment
can be written as:
</P>

<PRE>
    A11 A12 A13 .......... A1n
    A21 A22 A23 .......... A2n
    .
    .
    Am1 Am2 Am3 .......... Amn
</PRE>

<P>
We also have a residue comparison matrix of size R where C(i,j) is the score
for aligning residue i with residue j.
</P>

<P>
We calculate sequence weights by building a neighbour-joining tree, in which
branch lengths are proportional to divergence. Summing the branches by branch
ownership provides the weights. See (Thompson et al., CABIOS, 10, 19 (1994) and
Henikoff et al.,JMB, 243, 574 1994).
</P>

<P>
To find the low-scoring segments in a sequence Si, we build a weighted profile
of the remaining sequences in the alignment. Suppose we find residue r at 
position j in the sequence; then the score for the jth position in the sequence
is defined as
</P>

<PRE>
    Score(Si,j) = Profile(j,r)   where Profile(j,r) is the profile score
                                   for residue r at position j in the
                                   alignment.
</PRE>

<P>
These residue scores are summed along the sequence in both forward and backward
directions. If the sum of the scores is positive, then it is reset to zero.
Segments which score negatively in both directions are considered as 
'low-scoring' and will be highlighted in the alignment display.
</P>


>>HELP 9 <<
<H3>
              Command Line Parameters
</H3>

<H4>
                DATA (sequences)
</H4>

<PRE>
    -INFILE=file.ext                             :input sequences
    -PROFILE1=file.ext  and  -PROFILE2=file.ext  :profiles (aligned sequences)
</PRE>


<H4>
                VERBS (do things)
</H4>

<PRE>
    -OPTIONS          :list the command line parameters
    -HELP  or -CHECK  :outline the command line parameters
     (or  -FULLHELP)
    -ALIGN            :do full multiple alignment
    -TREE             :calculate NJ tree
    -BOOTSTRAP(=n)    :bootstrap a NJ tree (n= number of bootstraps; def. = 1000)
    -CONVERT          :output the input sequences in a different file format
</PRE>


<H4>
                PARAMETERS (set things)
</H4>

<H5>
General settings:
</H5>

<PRE>
    -INTERACTIVE   :read command line, then enter normal interactive menus
    -QUICKTREE     :use FAST algorithm for the alignment guide tree
    -TYPE=         :PROTEIN or DNA sequences
    -NEGATIVE      :protein alignment with negative values in matrix
    -OUTFILE=      :sequence alignment file name
    -OUTPUT=       :CLUSTAL, GCG, GDE, PHYLIP, PIR, NEXUS, FASTA
    -OUTORDER=     :INPUT or ALIGNED
    -CASE=         :LOWER or UPPER (for GDE output only)
    -SEQNOS=       :OFF or ON (for Clustal output only)
</PRE>


<H5>
Fast Pairwise Alignments:
</H5>

<PRE>
    -KTUPLE=n      :word size
    -TOPDIAGS=n    :number of best diags.
    -WINDOW=n      :window around best diags.
    -PAIRGAP=n     :gap penalty
    -SCORE=        :PERCENT or ABSOLUTE
</PRE>


<H5>
Slow Pairwise Alignments:
</H5>

<PRE>
    -PWMATRIX=     :Protein weight matrix=BLOSUM, PAM, GONNET, ID or filename
    -PWDNAMATRIX=  :DNA weight matrix=IUB, CLUSTALW or filename
    -PWGAPOPEN=f   :gap opening penalty
    -PWGAPEXT=f    :gap opening penalty
</PRE>
 

<H5>
Multiple Alignments:
</H5>

<PRE>
    -NEWTREE=      :file for new guide tree
    -USETREE=      :file for old guide tree
    -MATRIX=       :Protein weight matrix=BLOSUM, PAM, GONNET, ID or filename
    -DNAMATRIX=    :DNA weight matrix=IUB, CLUSTALW or filename
    -GAPOPEN=f     :gap opening penalty
    -GAPEXT=f      :gap extension penalty
    -ENDGAPS       :no end gap separation pen.
    -GAPDIST=n     :gap separation pen. range
    -NOPGAP        :residue-specific gaps off
    -NOHGAP        :hydrophilic gaps off
    -HGAPRESIDUES= :list hydrophilic res.
    -MAXDIV=n      :% ident. for delay
    -TYPE=         :PROTEIN or DNA
    -TRANSWEIGHT=f :transitions weighting
    -ITERATION=    :NONE or TREE or ALIGNMENT
    -NUMITER=n     :maximum number of iterations to perform
</PRE>


<H5>
Profile Alignments:
</H5>

<PRE>
    -PROFILE       :Merge two alignments by profile alignment
    -NEWTREE1=     :file for new guide tree for profile1
    -NEWTREE2=     :file for new guide tree for profile2
    -USETREE1=     :file for old guide tree for profile1
    -USETREE2=     :file for old guide tree for profile2
</PRE>


<H5>
Sequence to Profile Alignments:
</H5>

<PRE>
    -SEQUENCES     :Sequentially add profile2 sequences to profile1 alignment
    -NEWTREE=      :file for new guide tree
    -USETREE=      :file for old guide tree
</PRE>


<H5>
Structure Alignments:
</H5>

<PRE>
    -NOSECSTR1     :do not use secondary structure/gap penalty mask for profile 1 
    -NOSECSTR2     :do not use secondary structure/gap penalty mask for profile 2
    -SECSTROUT=STRUCTURE or MASK or BOTH or NONE  :output in alignment file
    -HELIXGAP=n    :gap penalty for helix core residues 
    -STRANDGAP=n   :gap penalty for strand core residues
    -LOOPGAP=n     :gap penalty for loop regions
    -TERMINALGAP=n :gap penalty for structure termini
    -HELIXENDIN=n  :number of residues inside helix to be treated as terminal
    -HELIXENDOUT=n :number of residues outside helix to be treated as terminal
    -STRANDENDIN=n :number of residues inside strand to be treated as terminal
    -STRANDENDOUT=n:number of residues outside strand to be treated as terminal 
</PRE>


<H5>
Trees:
</H5>

<PRE>
    -OUTPUTTREE=nj OR phylip OR dist OR nexus
    -SEED=n        :seed number for bootstraps
    -KIMURA        :use Kimura's correction
    -TOSSGAPS      :ignore positions with gaps
    -BOOTLABELS=node OR branch :position of bootstrap values in tree display
    -CLUSTERING=   :NJ or UPGMA
</PRE>


>>HELP R <<
<H3>
                             References
</H3>

<H4>
Version 2 of  ClustalW and ClustalX is described in:
</H4>

<P>
Larkin,M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A.,
McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R.,
Thompson, J.D., Gibson, T.J., Higgins, D.G. (2007)
Clustal W and Clustal X version 2.0. Bioinformatics, 23:2947-2948.
</P>


<H4>
The ClustalX program is described in:
</H4>

<P>
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,
25:4876-4882.
</P>


<H4>
The ClustalW program is described in:
</H4>

<P>
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.
</P>


<H4>
The ClustalV program is described in:
</H4>

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


<H4>
The original Clustal program is described in the manuscripts:
</H4>

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

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

<H4>
Some tips on using Clustal X:
</H4>

<P>
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.
</P>

<H4>
Some tips on using Clustal W:
</H4>

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

<H4>
You can get the latest version of the ClustalX program by anonymous ftp to:
</H4>

<PRE>
    ftp://ftp.ebi.ac.uk/pub/software/clustalw2
</PRE>

<H4>
Or, have a look at the following WWW sites:
</H4>

<PRE>
    http://www.clustal.org
    http://www.ebi.ac.uk/Tools/clustalw2/
</PRE>