clustalw_help

经典生物信息学多序列比对工具clustalw

Further help is offered in the weight matrix menu.


7)  In the weight matrices, 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.

8) PROTEIN GAP PARAMETERS displays a menu allowing you to set some Gap Penalty
options which are only used in protein alignments.

 
>>HELP A <<           Help for protein gap parameters.
1) 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.

2) 3) 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 are set by menu item 3.

4) 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.

5) 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.
>>HELP 5 <<      Help for output format options.

Six output formats are offered. You can choose any (or all 6 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 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 is the flat file format used by the GDE package of Steven Smith.

NEXUS: the format used by several phylogeny programs, including PAUP and
MacClade.

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, the order corresponds to the order in which the
sequences were aligned (from the guide tree-dendrogram), thus automatically
grouping closely related sequences. This switch can be used to set the order
to the same as the input file.

PARAMETER OUTPUT: This option allows you to save all your parameter settings
in a parameter file. This file can be used subsequently to rerun Clustal W
using the same parameters.

>>HELP 6 <<      Help for 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
W). 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. 

The profiles can be in any of the allowed input formats with "-" characters
used to specify gaps (except for MSF-RSF where "." is used).

You have to specify the 2 profiles by choosing menu items 1 and 2 and giving
2 file names.  Then Menu item 3 will align the 2 profiles to each other. 
Secondary structure masks in either profile can be used to guide the alignment.

Menu item 4 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 would not be pre-aligned.


The alignment parameters can be set using menu items 5, 6 and 7. 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.

SECONDARY STRUCTURE OPTIONS. Menu Option 0 allows you to set 2D structure
parameters. 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 loops.
Alternatively, a user-specified gap penalty mask can be supplied directly.

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 the help under option 0 (secondary structure options).
>>HELP B <<      Help for secondary structure - gap penalty masks

The use of secondary structure-based penalties has been shown to improve the
accuracy of multiple alignment. Therefore CLUSTAL W now allows gap penalty 
masks to be supplied with the input sequences. 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).

Options 1 and 2 control whether the input secondary structure information or
gap penalty masks will be used.

Option 3 controls whether the secondary structure and gap penalty masks should
be included in the output alignment.

Options 4 and 5 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. The basic gap penalties are
multiplied by the amount specified.

Option 6 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.

Option 7 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.

Options 8 and 9 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 W 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 W looks for SWISS-PROT HELIX and STRAND assignments e.g.

FT   HELIX       100    115
FT   STRAND      118    119

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.

>>HELP C <<      Help for secondary structure - gap penalty mask output options
   
   The options in this menu let you choose whether or not to include the masks
in the CLUSTAL W output alignments. Showing both is useful for understanding
how the masks work. The secondary structure information is itself very useful
in judging the alignment quality and in seeing how residue conservation
patterns vary with secondary structure. 


>>HELP 7 <<      Help for phylogenetic trees

1) Before calculating a tree, you must have an ALIGNMENT in memory. This can be
input in any format 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.

2) 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).  



3) CORRECT FOR MULTIPLE SUBSTITUTIONS? For small divergence (say <10%) this
option makes no difference. For greater divergence, it 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.  

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.  

4) To calculate a tree, use option 4 (DRAW TREE NOW). This gives an UNROOTED