Manual ClustalX PDF

ClustalX Help
You can get the latest version of the ClustalX program here:
ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/
For full details of usage and algorithms, please read the ClustalW.Doc file.
Toby Gibson EMBL, Heidelberg, Germany.
Des Higgins UCC, Cork, Ireland.
Julie Thompson/Francois Jeanmougin IGBMC, Strasbourg, France.

Index
General help for CLUSTAL X 1.
Input / Output Files 2.
Editing Alignments 3.
Multiple Alignments 4.
Profile and Structure Alignments 5.
Secondary Structure / Gap Penalty Masks 6.
Phylogenetic Trees 7.
Colors 8.
Alignment Quality Analysis 9.
Command Line Parameters 10.
References 11.
General help for CLUSTAL X

Clustal X is a new 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.
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.
Alignment quality analysis can be performed and low-scoring segments or exceptional residues can be highlighted.
ClustalX is available for a number of different platforms including: SUN Solaris, IRIX5.3 on Silicon Graphics,
Digital UNIX on DECStations, Microsoft Windows (32 bit) for PC's, Linux ELF for x86 PC's and Macintosh
PowerMac. (See the README file for Installation instructions.)

SEQUENCE INPUT
Sequences (and profiles) are input using the FILE menu. Invalid options will be disabled. All sequences must be in 1
file, one after another. 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 "-" which is used to indicate a GAP ("." in MSF/RSF).
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SEQUENCE / PROFILE ALIGNMENTS
Clustal X has two modes which can be selected using the switch directly above the sequence display: MULTIPLE
ALIGNMENT MODE and PROFILE ALIGNMENT MODE.
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.
In PROFILE ALIGNMENT MODE, two sequence data areas are displayed, allowing you to align 2 alignments (or
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 "-" character. PROFILES can be input in ANY
of the allowed formats; just use "-" (or "." 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.
PHYLOGENETIC TREES
Phylogenetic trees can be calculated from old alignments (read in with "-" characters to indicate gaps) OR after a
multiple alignment while the alignment is still displayed.
ALIGNMENT DISPLAY
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).
A ruler is displayed below the sequences, starting at 1 for the first residue position (residue numbers in the sequence
input file are ignored).
The line above the ruler is used to mark strongly conserved positions. Three characters ('*', ':' and '.') are 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

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

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Input / Output Files

LOAD SEQUENCES reads sequences from one of 7 file formats, replacing any sequences that are already loaded.
All sequences must be in 1 file, one after another. 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 "-" which is used to indicate a GAP
("." in MSF/RSF).
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.
FASTA and NBRF/PIR formats are recognised by having a ">" as the first character in the file.
EMBL/Swiss Prot formats are recognised by the letters ID at the start of the file (the token for the entry name field).
CLUSTAL format is recognised by the word CLUSTAL at the beginning of the file.
GCG/MSF format is recognised by one of the following:
- the word PileUp at the start of the file. G
- the word !!AA_MULTIPLE_ALIGNMENT or !!NA_MULTIPLE_ALIGNMENT at the start of the file. G
- the word MSF on the first line of the file, and the characters .. at the end of this line. G
GCG/RSF format is recognised by the word !!RICH_SEQUENCE at the beginning of the file.

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!
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.
SAVE SEQUENCES AS... offers the user a choice of one of five output formats: CLUSTAL, NBRF/PIR, GCG/MSF,
PHYLIP or GDE. All sequences are written to a single file. Options are available to switch between UPPER/LOWER
case for GDE files, and to output SEQUENCE NUMBERING for CLUSTAL files.
LOAD PROFILE 1 reads sequences in the same 6 file formats, replacing any sequences already loaded as Profile 1.
This option will also remove any sequences which are loaded in Profile 2.
LOAD PROFILE 2 reads sequences in the same 6 file formats, replacing any sequences already loaded as Profile 2.
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.
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.

WRITE ALIGNMENT AS POSTSCRIPT will write the sequence display to a postscript format file. This will include
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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.
WRITE PROFILE 1 AS POSTSCRIPT is similar to Write Alignment as Postscript except that only the profile 1
display will be printed.
WRITE PROFILE 2 AS POSTSCRIPT is similar to Write Alignment as Postscript except that only the profile 2
display will be printed.

POSTSCRIPT PARAMETERS
A number of options are available to allow you to configure your postscript output file.
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.
By default, Clustal X looks for a file called 'colprint.par' 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.
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,
RED 0.9 0.1 0.1
Blank lines and comments (lines beginning with a '#' character) are ignored.

PAGE SIZE: The alignment can be displayed on either A4 or A3 pages.
ORIENTATION: The alignment can be displayed on either a landscape or portrait page.
PRINT HEADER: An optional header including the postscript filename, and creation date can be printed at the top of
each page.
PRINT QUALITY CURVE: The Alignment Quality curve which is displayed underneath the alignment on the screen
can be included in the postscript output.
PRINT RULER: The ruler which is displayed underneath the alignment on the screen can be included in the
postscript output.
PRINT RESIDUE NUMBERS: Sequence residue numbers can be printed at the right hand side of the alignment.
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.
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.
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.
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ClustalX Help
Editing Alignments

Clustal X allows you to change the order of the sequences in the alignment, by cutting-and-pasting the sequence
names.
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.
(Options are provided to Select All Sequences, Select Profile 1 or Select Profile 2.)
The selected sequences can be removed from the alignment by using the EDIT menu, CUT option.
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.
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.
The sequence selection and sequence range selection can be cleared using the EDIT menu, CLEAR SEQUENCE
SELECTION and CLEAR RANGE SELECTION options respectively.
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.
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.
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 the most divergent sequences from an alignment.
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Multiple Alignments

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.
Multiple alignments are carried out in 3 stages:
1) all sequences are compared to each other (pairwise alignments);
2) a dendrogram (like a phylogenetic tree) is constructed, describing the approximate groupings of the sequences by
similarity (stored in a file).
3) the final multiple alignment is carried out, using the dendrogram as a guide.
The 3 stages are carried out automatically by the DO COMPLETE ALIGNMENT option. You can skip the first stage
(pairwise alignments; guide tree) by using an old guide tree file (DO ALIGNMENT FROM TREE); or you can just
produce the guide tree with no final multiple alignment (DO COMPLETE ALIGNMENT).

REALIGN SELECTED SEQUENCES is used to realign badly aligned sequences in the alignment. Sequences can be
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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.

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.
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.

ALIGNMENT PARAMETERS displays a sub-menu with the following options:
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.
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.

Pairwise Alignment parameters control the speed/sensitivity of the initial alignments.
Multiple Alignment parameters control the gaps in the final multiple alignments.
Protein Gap Parameters displays a temporary window which allows you to set various parameters only used in the
alignment of protein sequences.

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 .log appended.

OUTPUT FORMAT OPTIONS
You can choose from 5 different alignment formats (CLUSTAL, GCG, NBRF/PIR, PHYLIP and GDE). You can
choose more than one (or all 5 if you wish).
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 a new sequence with a profile
alignment.
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
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7 of GCG.
PHYLIP format output can be used for input to the PHYLIP package of Joe Felsenstein. This is an extremely widely
used package for doing every imaginable form of phylogenetic analysis (MUCH more than the the modest intro-
duction offered by this program).
NBRF/PIR: this is the same as the standard PIR format with ONE ADDITION. Gap characters "-" 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.
GDE: this format is used by the GDE package of Steven Smith.
GDE OUTPUT CASE: sequences in GDE format may be written in either upper or lower case.
CLUSTALW SEQUENCE NUMBERS: residue numbers may be added to the end of the alignment lines in clustalw
format.
OUTPUT ORDER is used to control the order of the sequences in the output alignments. By default, it is the same as
the input order. This switch can be used to make the order correspond to the order in which the sequences were
aligned (from the guide tree/dendrogram), thus automatically grouping closely related sequences.
PARAMETER OUTPUT: This option allows you to save all your parameter settings in a parameter file. This file can
be used subsequently to rerun ClustalW using the same parameters.

ALIGNMENT PARAMETERS

PAIRWISE ALIGNMENT PARAMETERS
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).
You can choose between the 2 alignment methods using the PAIRWISE ALIGNMENTS option. The slow/accurate
method is fine for short sequences but will be VERY SLOW for many (e.g. >20) long (e.g. >1000 residue) sequences.

SLOW/ACCURATE alignment parameters:
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.
Gap Open Penalty: the penalty for opening a gap in the alignment.
Gap Extension Penalty: the penalty for extending a gap by 1 residue.
Protein Weight Matrix: the scoring table which describes the similarity of each amino acid to each other. Load protein
matrix: allows to read in a comparison table from a file.
DNA weight matrix: the scores assigned to matches and mismatches (including IUB ambiguity codes). Load DNA
matrix: allows to read in a comparison table from a file.
See the Multiple alignment parameters, MATRIX option below for details of the matrix input format.

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FAST/APPROXIMATE alignment parameters:
These similarity scores are calculated from fast, approximate, global alignments, 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.

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

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 need to
increase the default.

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.

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

MULTIPLE ALIGNMENT PARAMETERS
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.
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.
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.
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.
The TRANSITION WEIGHT gives transitions (A <--> G or C <--> 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 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.
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
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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.
1) BLOSUM (Henikoff). These matrices appear to be the best available for carrying out data base similarity
(homology searches). The matrices used are: Blosum80, 62, 40 and 30.
2) PAM (Dayhoff). These have been extremely widely used since the late '70s. We use the PAM 120, 160, 250 and
350 matrices.
3) 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
use the GONNET 40, 80, 120, 160, 250 and 350 matrices.
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.
Load protein matrix: allows to read in a comparison matrix from a file. This can be either a single matrix or a series of
matrices.

DNA WEIGHT MATRIX option allows you to select the single matrix (not a series) used for aligning nucleic acid
sequences. Two hard-coded matrices are available:
1) 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.
2) 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.
Load DNA matrix: allows to read in a comparison matrix from a file (just one matrix, not a series).

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 # 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 * 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 * character) contain the minimum score over the whole matrix.
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).
Example.
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

PROTEIN GAP PARAMETERS

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
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that are rich in glycine are more likely to have an adjacent gap than positions that are rich in valine.
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.
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.
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.

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Profile and Structure Alignments

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.
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.
The profiles can be in any of the allowed input formats with "-" characters used to specify gaps (except for GCG/MSF
where "." is used).
You have to load the 2 profiles by choosing FILE, LOAD PROFILE 1 and LOAD 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.
You can skip the first stage (pairwise alignments; guide trees) by using an old guide tree file (ALIGN PROFILES
FROM GUIDE TREES).
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 need not be pre-aligned.
You can skip the first stage (pairwise alignments; guide tree) by using an old guide tree file (ALIGN SEQUENCES
TO PROFILE 1 FROM TREE).
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 .log appended.
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.
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SECONDARY STRUCTURE PARAMETERS
Use this menu to set secondary structure options. If a solved structure is available, 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.
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.
The format for gap penalty masks and secondary structure masks is explained in a separate section.
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Secondary Structure / Gap Penalty Masks

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).
The USE PROFILE 1/2 SECONDARY STRUCTURE / GAP PENALTY MASK options control whether the input
secondary structure information or gap penalty masks will be used during the profile alignment.
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 secondary structure
information is itself useful in judging the alignment quality and in seeing how residue conservation patterns vary with
secondary structure.
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.
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 "." in the secondary structure notation.
The SECONDARY STRUCTURE TERMINAL PENALTY provides the value for setting the gap penalty at the ends
of secondary structures. Ends of secondary structures are observed to grow and/or shrink in 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.
The HELIX and STRAND TERMINAL PENALTY 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. 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.
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.

FT HELIX 100 115
FT STRAND 118 119

ClustalX Help
The structure and penalty masks can also be read from CLUSTAL alignment format as comment lines beginning
"!SS_" or "!GM_" e.g.
!SS_HBA_HUMA ..aaaAAAAAAAAAAaaa.aaaAAAAAAAAAAaaaaaaAaaa.........aaaAAAAAA
!GM_HBA_HUMA 112224444444444222122244444444442222224222111111111222444444
HBA_HUMA VLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLSHGSAQVKGHGK

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.
In GDE flat file format, the masks are specified as text and the names must begin with SS_ or GM_.
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.
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.

Back to Index
Phylogenetic Trees

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!!!!
The method used is the NJ (Neighbour Joining) method of Saitou and Nei. First you calculate distances (percent
divergence) between all pairs of sequence from a multiple alignment; second you apply the NJ method to the distance
matrix.

To calculate a tree, use the DRAW N-J TREE option. This gives an UNROOTED tree and all branch lengths. 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).

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 very large number of bootstrap replicates (1000 is
recommended, even if it means running the program for an hour on a slow microcomputer; on a workstation it will be
MUCH faster). 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.

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. 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.
ClustalX Help

CORRECT FOR MULTIPLE SUBSTITUTIONS? For small divergence (say <10%) this option makes no difference.
For greater divergence, this option corrects for the fact that observed distances underestimate actual evolutionary dist-
ances. 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.
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.

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 .log appended.
OUTPUT FORMAT OPTIONS
Three 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.
1) Clustal format output. 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.
2) Phylip format output. 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.
3) The distances only. 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).
TOGGLE PHYLIP BOOTSTRAP POSITIONS By default, the bootstrap values are placed on the nodes of the phylip
format output tree. This is inaccurate as the bootstrap values should be associated with the tree branches and not the
nodes. However, this format can be read and displayed by TreeTool, TreeView and Phylowin. An option is available
to correctly place the bootstrap values on the branches with which they are associated.

Back to Index
Colors

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.
The 'rules' used to color the alignment are specified in a COLOR PARAMETER FILE. Clustal X automatically looks
ClustalX Help
for a file called 'colprot.par' for protein sequences or 'coldna.par' for DNA, 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).
By default, if no color parameter file is found, protein sequences are colored by residue as follows:
Color Residue Code

ORANGE GPST
RED HKR
BLUE FWY
GREEN ILMV

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

ORANGE A
RED C
BLUE T
GREEN G

The default coloring system is to show residues as a colored character on a white background. The BACKGROUND
COLORING option shows the sequence residues using a black character on a colored background.
The DEFAULT COLOR PARAMETERS option looks first for a file called color.par (as described above) and, if no
file is found, uses the default residue-specific colors.
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.
COLOR PARAMETER FILE
This file is divided into 3 sections:
1) the names and rgb values of the colors 2) the rules for calculating the consensus 3) the rules for assigning colors to
the residues
An example file is given here.
--------------------------------------------------------------------
@rgbindex
RED 0.9 0.1 0.1
BLUE 0.1 0.1 0.9
GREEN 0.1 0.9 0.1
YELLOW 0.9 0.9 0.0

@consensus
% = 60% w:l:v:i:m:a:f:c:y:h:p
# = 80% w:l:v:i:m:a:f:c:y:h:p
- = 50% e:d
+ = 60% k:r
q = 50% q:e
p = 50% p
n = 50% n
t = 50% t:s
ClustalX Help

@color
g = RED
p = YELLOW
t = GREEN if t:%:#
n = GREEN if n
w = BLUE if %:#:p
k = RED if +
--------------------------------------------------------------------

The first section is optional and is identified by the header @rgbindex. If this section exists, each color used in the file
must be named and the rgb values specified (on a scale from 0 to 1). If the rgb index section is not found, the
following set of hard-coded colors will be used.
RED 0.9 0.1 0.1
BLUE 0.1 0.1 0.9
GREEN 0.1 0.9 0.1
ORANGE 0.9 0.7 0.3
CYAN 0.1 0.9 0.9
PINK 0.9 0.5 0.5
MAGENTA 0.9 0.1 0.9
YELLOW 0.9 0.9 0.0

The second section is optional and is identified by the header @consensus. It defines how the consensus is calculated.
The format of each consensus parameter is:-
c = n% residue_list

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 denoted by a single
character, delimited by a colon (:).

For example: # = 60% w:l:v:i
will assign a consensus character # to any column in the alignment which contains more than 60% of the residues
w,l,v and i.

The third section is identified by the header @color, and defines how colors are assigned to each residue in the
alignment.
The color parameters can take one of two formats:
1) r = color
2) r = color if consensus_list

where
r is a character used to denote a residue.
ClustalX Help
color is one of the colors in the GDE color lookup table.
residue_list is a list of residues denoted by a single
character, delimited by a colon (:).

Examples: 1) g = ORANGE
will color all glycines ORANGE, regardless of the consensus.
2) w = BLUE if w:%:#
will color BLUE any tryptophan which is found in a column with a consensus of w, % or #.

Back to Index
Alignment Quality Analysis

QUALITY SCORES
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 sequences / profiles.
Two methods are also provided to indicate single residues or sequence segments which score badly in the alignment.
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:
1. Partial or total misalignments caused by a failure in the alignment algorithm. Usually only in difficult alignment
cases.
2. 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.
3. 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.
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.

SHOW RESIDUE EXCEPTIONS
An option is available to highlight 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.
For details of the quality score calculations, see the CALCULATION section below.

QUALITY SCORE PARAMETERS
ClustalX Help
You can customise the 'quality scores' plotted underneath the alignment display using the following options.
SCORE SCALE: this is a scalar value from 1 to 10, which can be used to change the scale of the quality score plot.
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.)

PROTEIN WEIGHT MATRIX: the scoring table which describes the similarity of each amino acid to each other.
DNA WEIGHT MATRIX: two hard-coded matrices are available: IUB and CLUSTALW(1.6).
For more information about the weight matrices, see the help above for the Low-scoring Segments Weight Matrix.

LOW-SCORING SEGMENTS
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.
For details of the low-scoring segment calculation, see the CALCULATION section below.

LOW-SCORING SEGMENT PARAMETERS
MINIMUM LENGTH OF SEGMENTS: short segments (or even single residues) can be hidden by increasing the
minimum length of segments which will be displayed.
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.
HIDE LOW-SCORING SEGMENTS: The segment display can be toggled on or off. This option does not recalculate
the profile scores.

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' weight matrices offered: the
Blosum 45 matrix and the Gonnet PAM 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.

DNA WEIGHT MATRIX: Two hard-coded matrices are available:
1) 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.
2) 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. For DNA, a single hard-coded matrix is used. 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 0.0.
ClustalX Help
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.
INPUT FORMAT The format used for a new matrix is the same as the BLAST program. Any lines beginning with a
# 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 * 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 * character)
contain the minimum score over the whole matrix.
CALCULATION OF QUALITY SCORES
Suppose we have an alignment of m sequences of length n. Then, the alignment can be written as:
A11 A12 A13 .......... A1n
A21 A22 A23 .......... A2n
.
.
Am1 Am2 Am3 .......... Amn

We also have a residue comparison matrix of size R where C(i,j) is the score for aligning residue i with residue j.
We want to calculate a score for the conservation of the jth position in the alignment.
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:
Sr = C(r,Aij)

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:
Xr = ( SUM (Fij * C(i,r)) ) / m
1<=i<=R

where Fij is the count of residues i at position j in the alignment.
Now we can calculate the distance Di between each sequence i and the consensus position X in the R-dimensional
space.
Di = SQRT ( SUM (Xr - Sr)(Xr - Sr) )
1<=i<=R

The quality score for the jth position in the alignment is defined as the mean of the sequence distances Di.
The score is normalised by multiplying by the percentage of sequences which have residues (and not gaps) at this
position.
CALCULATION OF RESIDUE EXCEPTIONS
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.
(NB. Sequences which contain gaps at this position are not included in the exception calculation.)
ClustalX Help

CALCULATION OF LOW-SCORING SEGMENTS
Suppose we have an alignment of m sequences of length n. Then, the alignment can be written as:
A11 A12 A13 .......... A1n
A21 A22 A23 .......... A2n
.
.
Am1 Am2 Am3 .......... Amn

We also have a residue comparison matrix of size R where C(i,j) is the score for aligning residue i with residue j.
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).
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
Score(Si,j) = Profile(j,r) where Profile(j,r) is the profile score
for residue r at position j in the
alignment.

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.

Back to Index
Command Line Parameters
DATA (sequences)
Parameter Description
/PROFILE1=file.ext and /PROFILE2=file.ext profiles (aligned sequences).
VERBS (do things)
/HELP or /CHECK outline the command line params.
/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.
ClustalX Help
PARAMETERS (set things)
***General settings:****
/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= GCG, GDE, PHYLIP or PIR
/OUTORDER= INPUT or ALIGNED
/CASE= LOWER or UPPER (for GDE output only)
/SEQNOS= OFF or ON (for Clustal output only)
***Fast Pairwise Alignments:***
/TOPDIAGS=n number of best diags.
/WINDOW=n window around best diags.
/PAIRGAP=n gap penalty
/SCORE= PERCENT or ABSOLUTE
***Slow Pairwise Alignments:***
/PWDNAMATRIX= DNA weight matrix=IUB, CLUSTALW or filename
/PWGAPOPEN=f gap opening penalty
/PWGAPEXT=f gap opening penalty
***Multiple Alignments:***
/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
ClustalX Help
/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
***Profile Alignments:***
/SEQUENCES Sequentially add profile2 sequences to profile1 alignment
/NEWTREE1= file for new guide tree for profile 1
/USETREE1= file for old guide tree for profile 1
/NEWTREE2= file for new guide tree for profile 2
/USETREE2= file for old guide tree for profile 2
***Structure Alignments:***
/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
ClustalX Help
***Trees:***
/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.
Back to Index
References

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

The ClustalW program is described in the manuscript:
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.

The ClustalV program is described in the manuscript:
Higgins,D.G., Bleasby,A.J. and Fuchs,R. (1992) CLUSTAL V: improved software for multiple sequence alignment.
CABIOS 8,189-191.

The original Clustal program is described in the manuscripts:
Higgins,D.G. and Sharp,P.M. (1989) Fast and sensitive multiple sequence alignments on a microcomputer. CABIOS
5,151-153.
Higgins,D.G. and Sharp,P.M. (1988) CLUSTAL: a package for performing multiple sequence alignment on a
microcomputer. Gene 73,237-244.

Some tips on using Clustal W:
Higgins, D. G., Thompson, J. D. and Gibson, T. J. (1996) Using CLUSTAL for multiple sequence alignments.
Methods Enzymol., 266, 383-402.

You can get the latest version of the ClustalX program by anonymous ftp to:
ftp-igbmc.u-strasbg.fr ftp.embl-heidelberg.de ftp.ebi.ac.uk
Or, have a look at the following WWW site:
ClustalX Help
http://www-igbmc.u-strasbg.fr/BioInfo/
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