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1
2This is the on-line help file for CLUSTAL W ( version 1.83).   
3
4It should be named or defined as: clustalw_help
5except with MSDOS in which case it should be named CLUSTALW.HLP
6
7For full details of usage and algorithms, please read the CLUSTALW.DOC file.
8
9
10Toby  Gibson                         EMBL, Heidelberg, Germany.
11Des   Higgins                        UCC, Cork, Ireland.
12Julie Thompson                       IGBMC, Strasbourg, France.
13
14
15
16>>NEW <<
17
18  Fasta output
19  ===========
20
21  Write/Read sequence with range specified. The command line syntax
22   for range specification is flexible. You can use one of the following
23   syntax.
24
25       -range=n:m 
26       -range=n-m
27       -range="n m"
28
29   where m is the starting and m is the length of the sequence.
30
31  Range and range numbers.
32  =======================
33
34  Include range numbers in the ouput.
35
36       -seqno_range=on/off
37
38  The sequence range will be appended as to the names of the sequence.
39
40
41  PIM: Percentage Identity Matrix
42  ===============================
43
44
45
46>>HELP 1 <<             General help for CLUSTAL W (1.81)
47
48Clustal W is a general purpose multiple alignment program for DNA or proteins.
49
50SEQUENCE INPUT:  all sequences must be in 1 file, one after another. 
517 formats are automatically recognised: NBRF-PIR, EMBL-SWISSPROT,
52Pearson (Fasta), Clustal (*.aln), GCG-MSF (Pileup), GCG9-RSF and GDE flat file.
53All non-alphabetic characters (spaces, digits, punctuation marks) are ignored
54except "-" which is used to indicate a GAP ("." in MSF-RSF). 
55
56To do a MULTIPLE ALIGNMENT on a set of sequences, use item 1 from this menu to
57INPUT them; go to menu item 2 to do the multiple alignment.
58
59PROFILE ALIGNMENTS (menu item 3) are used to align 2 alignments.  Use this to
60add a new sequence to an old alignment, or to use secondary structure to guide
61the alignment process.  GAPS in the old alignments are indicated using the "-"
62character.   PROFILES can be input in ANY of the allowed formats; just
63use "-" (or "." for MSF-RSF) for each gap position.
64
65PHYLOGENETIC TREES (menu item 4) can be calculated from old alignments (read in
66with "-" characters to indicate gaps) OR after a multiple alignment while the
67alignment is still in memory.
68
69
70The program tries to automatically recognise the different file formats used
71and to guess whether the sequences are amino acid or nucleotide.  This is not
72always foolproof.
73
74FASTA and NBRF-PIR formats are recognised by having a ">" as the first
75character in the file. 
76
77EMBL-Swiss Prot formats are recognised by the letters
78ID at the start of the file (the token for the entry name field). 
79
80CLUSTAL format is recognised by the word CLUSTAL at the beginning of the file.
81
82GCG-MSF format is recognised by one of the following:
83       - the word PileUp at the start of the file.
84       - the word !!AA_MULTIPLE_ALIGNMENT or !!NA_MULTIPLE_ALIGNMENT
85         at the start of the file.
86       - the word MSF on the first line of the line, and the characters ..
87         at the end of this line.
88
89GCG-RSF format is recognised by the word !!RICH_SEQUENCE at the beginning of
90the file.
91
92
93If 85% or more of the characters in the sequence are from A,C,G,T,U or N, the
94sequence will be assumed to be nucleotide.  This works in 97.3% of cases
95but watch out!
96
97>>HELP 2 <<      Help for multiple alignments
98
99If you have already loaded sequences, use menu item 1 to do the complete
100multiple alignment.  You will be prompted for 2 output files: 1 for the
101alignment itself; another to store a dendrogram that describes the similarity
102of the sequences to each other.
103
104Multiple alignments are carried out in 3 stages (automatically done from menu
105item 1 ...Do complete multiple alignments now):
106
1071) all sequences are compared to each other (pairwise alignments);
108
1092) a dendrogram (like a phylogenetic tree) is constructed, describing the
110approximate groupings of the sequences by similarity (stored in a file).
111
1123) the final multiple alignment is carried out, using the dendrogram as a guide.
113
114
115PAIRWISE ALIGNMENT parameters control the speed-sensitivity of the initial
116alignments.
117
118MULTIPLE ALIGNMENT parameters control the gaps in the final multiple alignments.
119
120
121RESET GAPS (menu item 7) will remove any new gaps introduced into the sequences
122during multiple alignment if you wish to change the parameters and try again.
123This only takes effect just before you do a second multiple alignment.  You
124can make phylogenetic trees after alignment whether or not this is ON.
125If you turn this OFF, the new gaps are kept even if you do a second multiple
126alignment. This allows you to iterate the alignment gradually.  Sometimes, the
127alignment is improved by a second or third pass.
128
129SCREEN DISPLAY (menu item 8) can be used to send the output alignments to the
130screen as well as to the output file.
131
132You can skip the first stages (pairwise alignments; dendrogram) by using an
133old dendrogram file (menu item 3); or you can just produce the dendrogram
134with no final multiple alignment (menu item 2).
135
136
137OUTPUT FORMAT: Menu item 9 (format options) allows you to choose from 6
138different alignment formats (CLUSTAL, GCG, NBRF-PIR, PHYLIP, GDE, NEXUS, and FASTA). 
139
140
141>>HELP 3 <<      Help for pairwise alignment parameters
142A distance is calculated between every pair of sequences and these are used to
143construct the dendrogram which guides the final multiple alignment. The scores
144are calculated from separate pairwise alignments. These can be calculated using
1452 methods: dynamic programming (slow but accurate) or by the method of Wilbur
146and Lipman (extremely fast but approximate).
147
148You can choose between the 2 alignment methods using menu option 8.  The
149slow-accurate method is fine for short sequences but will be VERY SLOW for
150many (e.g. >100) long (e.g. >1000 residue) sequences.   
151
152SLOW-ACCURATE alignment parameters:
153        These parameters do not have any affect on the speed of the alignments.
154They are used to give initial alignments which are then rescored to give percent
155identity scores.  These % scores are the ones which are displayed on the
156screen.  The scores are converted to distances for the trees.
157
1581) Gap Open Penalty:      the penalty for opening a gap in the alignment.
1592) Gap extension penalty: the penalty for extending a gap by 1 residue.
1603) Protein weight matrix: the scoring table which describes the similarity
161                          of each amino acid to each other.
1624) DNA weight matrix:     the scores assigned to matches and mismatches
163                          (including IUB ambiguity codes).
164
165
166FAST-APPROXIMATE alignment parameters:
167
168These similarity scores are calculated from fast, approximate, global align-
169ments, which are controlled by 4 parameters.   2 techniques are used to make
170these alignments very fast: 1) only exactly matching fragments (k-tuples) are
171considered; 2) only the 'best' diagonals (the ones with most k-tuple matches)
172are used.
173
174K-TUPLE SIZE:  This is the size of exactly matching fragment that is used.
175INCREASE for speed (max= 2 for proteins; 4 for DNA), DECREASE for sensitivity.
176For longer sequences (e.g. >1000 residues) you may need to increase the default.
177
178GAP PENALTY:   This is a penalty for each gap in the fast alignments.  It has
179little affect on the speed or sensitivity except for extreme values.
180
181TOP DIAGONALS: The number of k-tuple matches on each diagonal (in an imaginary
182dot-matrix plot) is calculated.  Only the best ones (with most matches) are
183used in the alignment.  This parameter specifies how many.  Decrease for speed;
184increase for sensitivity.
185
186WINDOW SIZE:  This is the number of diagonals around each of the 'best'
187diagonals that will be used.  Decrease for speed; increase for sensitivity.
188
189
190>>HELP 4 <<      Help for multiple alignment parameters
191
192These parameters control the final multiple alignment. This is the core of the
193program and the details are complicated. To fully understand the use of the
194parameters and the scoring system, you will have to refer to the documentation.
195
196Each step in the final multiple alignment consists of aligning two alignments
197or sequences.  This is done progressively, following the branching order in
198the GUIDE TREE.  The basic parameters to control this are two gap penalties and
199the scores for various identical-non-identical residues. 
200
2011) and 2) The GAP PENALTIES are set by menu items 1 and 2. These control the
202cost of opening up every new gap and the cost of every item in a gap.
203Increasing the gap opening penalty will make gaps less frequent. Increasing
204the gap extension penalty will make gaps shorter. Terminal gaps are not
205penalised.
206
2073) The DELAY DIVERGENT SEQUENCES switch delays the alignment of the most
208distantly related sequences until after the most closely related sequences have
209been aligned.   The setting shows the percent identity level required to delay
210the addition of a sequence; sequences that are less identical than this level
211to any other sequences will be aligned later.
212
213
214
2154) The TRANSITION WEIGHT gives transitions (A <--> G or C <--> T
216i.e. purine-purine or pyrimidine-pyrimidine substitutions) a weight between 0
217and 1; a weight of zero means that the transitions are scored as mismatches,
218while a weight of 1 gives the transitions the match score. For distantly related
219DNA sequences, the weight should be near to zero; for closely related sequences
220it can be useful to assign a higher score.
221
222
2235) PROTEIN WEIGHT MATRIX leads to a new menu where you are offered a choice of
224weight matrices. The default for proteins in version 1.8 is the PAM series
225derived by Gonnet and colleagues. Note, a series is used! The actual matrix
226that is used depends on how similar the sequences to be aligned at this
227alignment step are. Different matrices work differently at each evolutionary
228distance.
229
2306) DNA WEIGHT MATRIX leads to a new menu where a single matrix (not a series)
231can be selected. The default is the matrix used by BESTFIT for comparison of
232nucleic acid sequences.
233
234Further help is offered in the weight matrix menu.
235
236
2377)  In the weight matrices, you can use negative as well as positive values if
238you wish, although the matrix will be automatically adjusted to all positive
239scores, unless the NEGATIVE MATRIX option is selected.
240
2418) PROTEIN GAP PARAMETERS displays a menu allowing you to set some Gap Penalty
242options which are only used in protein alignments.
243
244 
245>>HELP A <<           Help for protein gap parameters.
2461) RESIDUE SPECIFIC PENALTIES are amino acid specific gap penalties that reduce
247or increase the gap opening penalties at each position in the alignment or
248sequence.  See the documentation for details.  As an example, positions that
249are rich in glycine are more likely to have an adjacent gap than positions that
250are rich in valine.
251
2522) 3) HYDROPHILIC GAP PENALTIES are used to increase the chances of a gap within
253a run (5 or more residues) of hydrophilic amino acids; these are likely to
254be loop or random coil regions where gaps are more common.  The residues that
255are "considered" to be hydrophilic are set by menu item 3.
256
2574) GAP SEPARATION DISTANCE tries to decrease the chances of gaps being too
258close to each other. Gaps that are less than this distance apart are penalised
259more than other gaps. This does not prevent close gaps; it makes them less
260frequent, promoting a block-like appearance of the alignment.
261
2625) END GAP SEPARATION treats end gaps just like internal gaps for the purposes
263of avoiding gaps that are too close (set by GAP SEPARATION DISTANCE above).
264If you turn this off, end gaps will be ignored for this purpose.  This is
265useful when you wish to align fragments where the end gaps are not biologically
266meaningful.
267>>HELP 5 <<      Help for output format options.
268
269Six output formats are offered. You can choose any (or all 6 if you wish). 
270
271CLUSTAL format output is a self explanatory alignment format.  It shows the
272sequences aligned in blocks.  It can be read in again at a later date to
273(for example) calculate a phylogenetic tree or add a new sequence with a
274profile alignment.
275
276GCG output can be used by any of the GCG programs that can work on multiple
277alignments (e.g. PRETTY, PROFILEMAKE, PLOTALIGN).  It is the same as the GCG
278.msf format files (multiple sequence file); new in version 7 of GCG.
279
280PHYLIP format output can be used for input to the PHYLIP package of Joe
281Felsenstein.  This is an extremely widely used package for doing every
282imaginable form of phylogenetic analysis (MUCH more than the the modest intro-
283duction offered by this program).
284
285NBRF-PIR:  this is the same as the standard PIR format with ONE ADDITION.  Gap
286characters "-" are used to indicate the positions of gaps in the multiple
287alignment.  These files can be re-used as input in any part of clustal that
288allows sequences (or alignments or profiles) to be read in. 
289
290GDE:  this is the flat file format used by the GDE package of Steven Smith.
291
292NEXUS: the format used by several phylogeny programs, including PAUP and
293MacClade.
294
295GDE OUTPUT CASE: sequences in GDE format may be written in either upper or
296lower case.
297
298CLUSTALW SEQUENCE NUMBERS: residue numbers may be added to the end of the
299alignment lines in clustalw format.
300
301OUTPUT ORDER is used to control the order of the sequences in the output
302alignments.  By default, the order corresponds to the order in which the
303sequences were aligned (from the guide tree-dendrogram), thus automatically
304grouping closely related sequences. This switch can be used to set the order
305to the same as the input file.
306
307PARAMETER OUTPUT: This option allows you to save all your parameter settings
308in a parameter file. This file can be used subsequently to rerun Clustal W
309using the same parameters.
310
311>>HELP 6 <<      Help for profile and structure alignments
312   
313By PROFILE ALIGNMENT, we mean alignment using existing alignments. Profile
314alignments allow you to store alignments of your favourite sequences and add
315new sequences to them in small bunches at a time. A profile is simply an
316alignment of one or more sequences (e.g. an alignment output file from CLUSTAL
317W). Each input can be a single sequence. One or both sets of input sequences
318may include secondary structure assignments or gap penalty masks to guide the
319alignment.
320
321The profiles can be in any of the allowed input formats with "-" characters
322used to specify gaps (except for MSF-RSF where "." is used).
323
324You have to specify the 2 profiles by choosing menu items 1 and 2 and giving
3252 file names.  Then Menu item 3 will align the 2 profiles to each other.
326Secondary structure masks in either profile can be used to guide the alignment.
327
328Menu item 4 will take the sequences in the second profile and align them to
329the first profile, 1 at a time.  This is useful to add some new sequences to
330an existing alignment, or to align a set of sequences to a known structure. 
331In this case, the second profile would not be pre-aligned.
332
333
334The alignment parameters can be set using menu items 5, 6 and 7. These are
335EXACTLY the same parameters as used by the general, automatic multiple
336alignment procedure. The general multiple alignment procedure is simply a
337series of profile alignments. Carrying out a series of profile alignments on
338larger and larger groups of sequences, allows you to manually build up a
339complete alignment, if necessary editing intermediate alignments.
340
341SECONDARY STRUCTURE OPTIONS. Menu Option 0 allows you to set 2D structure
342parameters. If a solved structure is available, it can be used to guide the
343alignment by raising gap penalties within secondary structure elements, so
344that gaps will preferentially be inserted into unstructured surface loops.
345Alternatively, a user-specified gap penalty mask can be supplied directly.
346
347A gap penalty mask is a series of numbers between 1 and 9, one per position in
348the alignment. Each number specifies how much the gap opening penalty is to be
349raised at that position (raised by multiplying the basic gap opening penalty
350by the number) i.e. a mask figure of 1 at a position means no change
351in gap opening penalty; a figure of 4 means that the gap opening penalty is
352four times greater at that position, making gaps 4 times harder to open.
353
354The format for gap penalty masks and secondary structure masks is explained
355in the help under option 0 (secondary structure options).
356>>HELP B <<      Help for secondary structure - gap penalty masks
357
358The use of secondary structure-based penalties has been shown to improve the
359accuracy of multiple alignment. Therefore CLUSTAL W now allows gap penalty
360masks to be supplied with the input sequences. The masks work by raising gap
361penalties in specified regions (typically secondary structure elements) so that
362gaps are preferentially opened in the less well conserved regions (typically
363surface loops).
364
365Options 1 and 2 control whether the input secondary structure information or
366gap penalty masks will be used.
367
368Option 3 controls whether the secondary structure and gap penalty masks should
369be included in the output alignment.
370
371Options 4 and 5 provide the value for raising the gap penalty at core Alpha
372Helical (A) and Beta Strand (B) residues. In CLUSTAL format, capital residues
373denote the A and B core structure notation. The basic gap penalties are
374multiplied by the amount specified.
375
376Option 6 provides the value for the gap penalty in Loops. By default this
377penalty is not raised. In CLUSTAL format, loops are specified by "." in the
378secondary structure notation.
379
380Option 7 provides the value for setting the gap penalty at the ends of
381secondary structures. Ends of secondary structures are observed to grow
382and-or shrink in related structures. Therefore by default these are given
383intermediate values, lower than the core penalties. All secondary structure
384read in as lower case in CLUSTAL format gets the reduced terminal penalty.
385
386Options 8 and 9 specify the range of structure termini for the intermediate
387penalties. In the alignment output, these are indicated as lower case.
388For Alpha Helices, by default, the range spans the end helical turn. For
389Beta Strands, the default range spans the end residue and the adjacent loop
390residue, since sequence conservation often extends beyond the actual H-bonded
391Beta Strand.
392
393CLUSTAL W can read the masks from SWISS-PROT, CLUSTAL or GDE format input
394files. For many 3-D protein structures, secondary structure information is
395recorded in the feature tables of SWISS-PROT database entries. You should
396always check that the assignments are correct - some are quite inaccurate.
397CLUSTAL W looks for SWISS-PROT HELIX and STRAND assignments e.g.
398
399FT   HELIX       100    115
400FT   STRAND      118    119
401
402The structure and penalty masks can also be read from CLUSTAL alignment format
403as comment lines beginning "!SS_" or "!GM_" e.g.
404
405!SS_HBA_HUMA    ..aaaAAAAAAAAAAaaa.aaaAAAAAAAAAAaaaaaaAaaa.........aaaAAAAAA
406!GM_HBA_HUMA    112224444444444222122244444444442222224222111111111222444444
407HBA_HUMA        VLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLSHGSAQVKGHGK
408
409Note that the mask itself is a set of numbers between 1 and 9 each of which is
410assigned to the residue(s) in the same column below.
411
412In GDE flat file format, the masks are specified as text and the names must
413begin with "SS_ or "GM_.
414
415Either a structure or penalty mask or both may be used. If both are included in
416an alignment, the user will be asked which is to be used.
417
418>>HELP C <<      Help for secondary structure - gap penalty mask output options
419   
420   The options in this menu let you choose whether or not to include the masks
421in the CLUSTAL W output alignments. Showing both is useful for understanding
422how the masks work. The secondary structure information is itself very useful
423in judging the alignment quality and in seeing how residue conservation
424patterns vary with secondary structure.
425
426
427>>HELP 7 <<      Help for phylogenetic trees
428
4291) Before calculating a tree, you must have an ALIGNMENT in memory. This can be
430input in any format or you should have just carried out a full multiple
431alignment and the alignment is still in memory.
432
433
434*************** Remember YOU MUST ALIGN THE SEQUENCES FIRST!!!! ***************
435
436
437The method used is the NJ (Neighbour Joining) method of Saitou and Nei. First
438you calculate distances (percent divergence) between all pairs of sequence from
439a multiple alignment; second you apply the NJ method to the distance matrix.
440
4412) EXCLUDE POSITIONS WITH GAPS? With this option, any alignment positions where
442ANY of the sequences have a gap will be ignored. This means that 'like' will be
443compared to 'like' in all distances, which is highly desirable. It also
444automatically throws away the most ambiguous parts of the alignment, which are
445concentrated around gaps (usually). The disadvantage is that you may throw away
446much of the data if there are many gaps (which is why it is difficult for us to
447make it the default). 
448
449
450
4513) CORRECT FOR MULTIPLE SUBSTITUTIONS? For small divergence (say <10%) this
452option makes no difference. For greater divergence, it corrects for the fact
453that observed distances underestimate actual evolutionary distances. This is
454because, as sequences diverge, more than one substitution will happen at many
455sites. However, you only see one difference when you look at the present day
456sequences. Therefore, this option has the effect of stretching branch lengths
457in trees (especially long branches). The corrections used here (for DNA or
458proteins) are both due to Motoo Kimura. See the documentation for details. 
459
460Where possible, this option should be used. However, for VERY divergent
461sequences, the distances cannot be reliably corrected. You will be warned if
462this happens. Even if none of the distances in a data set exceed the reliable
463threshold, if you bootstrap the data, some of the bootstrap distances may
464randomly exceed the safe limit. 
465
4664) To calculate a tree, use option 4 (DRAW TREE NOW). This gives an UNROOTED
467tree and all branch lengths. The root of the tree can only be inferred by
468using an outgroup (a sequence that you are certain branches at the outside
469of the tree .... certain on biological grounds) OR if you assume a degree
470of constancy in the 'molecular clock', you can place the root in the 'middle'
471of the tree (roughly equidistant from all tips).
472
4735) TOGGLE PHYLIP BOOTSTRAP POSITIONS
474By default, the bootstrap values are correctly placed on the tree branches of
475the phylip format output tree. The toggle allows them to be placed on the
476nodes, which is incorrect, but some display packages (e.g. TreeTool, TreeView
477and Phylowin) only support node labelling but not branch labelling. Care
478should be taken to note which branches and labels go together.
479
4806) OUTPUT FORMATS: four different formats are allowed. None of these displays
481the tree visually. Useful display programs accepting PHYLIP format include
482NJplot (from Manolo Gouy and supplied with Clustal W), TreeView (Mac-PC), and
483PHYLIP itself - OR get the PHYLIP package and use the tree drawing facilities
484there. (Get the PHYLIP package anyway if you are interested in trees). The
485NEXUS format can be read into PAUP or MacClade.
486
487>>HELP 8 <<      Help for choosing a weight matrix
488
489For protein alignments, you use a weight matrix to determine the similarity of
490non-identical amino acids.  For example, Tyr aligned with Phe is usually judged
491to be 'better' than Tyr aligned with Pro.
492
493There are three 'in-built' series of weight matrices offered. Each consists of
494several matrices which work differently at different evolutionary distances. To
495see the exact details, read the documentation. Crudely, we store several
496matrices in memory, spanning the full range of amino acid distance (from almost
497identical sequences to highly divergent ones). For very similar sequences, it
498is best to use a strict weight matrix which only gives a high score to
499identities and the most favoured conservative substitutions. For more divergent
500sequences, it is appropriate to use "softer" matrices which give a high score
501to many other frequent substitutions.
502
5031) BLOSUM (Henikoff). These matrices appear to be the best available for
504carrying out database similarity (homology searches). The matrices used are:
505Blosum 80, 62, 45 and 30. (BLOSUM was the default in earlier Clustal W
506versions)
507
5082) PAM (Dayhoff). These have been extremely widely used since the late '70s.
509We use the PAM 20, 60, 120 and 350 matrices.
510
5113) GONNET. These matrices were derived using almost the same procedure as the
512Dayhoff one (above) but are much more up to date and are based on a far larger
513data set. They appear to be more sensitive than the Dayhoff series. We use the
514GONNET 80, 120, 160, 250 and 350 matrices. This series is the default for
515Clustal W version 1.8.
516
517We also supply an identity matrix which gives a score of 1.0 to two identical
518amino acids and a score of zero otherwise. This matrix is not very useful.
519Alternatively, you can read in your own (just one matrix, not a series).
520
521A new matrix can be read from a file on disk, if the filename consists only
522of lower case characters. The values in the new weight matrix must be integers
523and the scores should be similarities. You can use negative as well as positive
524values if you wish, although the matrix will be automatically adjusted to all
525positive scores.
526
527
528
529For DNA, a single matrix (not a series) is used. Two hard-coded matrices are
530available:
531
532
5331) IUB. This is the default scoring matrix used by BESTFIT for the comparison
534of nucleic acid sequences. X's and N's are treated as matches to any IUB
535ambiguity symbol. All matches score 1.9; all mismatches for IUB symbols score 0.
536 
537 
5382) CLUSTALW(1.6). The previous system used by Clustal W, in which matches score
5391.0 and mismatches score 0. All matches for IUB symbols also score 0.
540
541INPUT FORMAT  The format used for a new matrix is the same as the BLAST program.
542Any lines beginning with a # character are assumed to be comments. The first
543non-comment line should contain a list of amino acids in any order, using the
5441 letter code, followed by a * character. This should be followed by a square
545matrix of integer scores, with one row and one column for each amino acid. The
546last row and column of the matrix (corresponding to the * character) contain
547the minimum score over the whole matrix.
548
549>>HELP 9 <<      Help for command line parameters
550                DATA (sequences)
551
552-INFILE=file.ext                             :input sequences.
553-PROFILE1=file.ext  and  -PROFILE2=file.ext  :profiles (old alignment).
554
555
556                VERBS (do things)
557
558-OPTIONS            :list the command line parameters
559-HELP  or -CHECK    :outline the command line params.
560-ALIGN              :do full multiple alignment
561-TREE               :calculate NJ tree.
562-BOOTSTRAP(=n)      :bootstrap a NJ tree (n= number of bootstraps; def. = 1000).
563-CONVERT            :output the input sequences in a different file format.
564
565
566                PARAMETERS (set things)
567
568***General settings:****
569-INTERACTIVE :read command line, then enter normal interactive menus
570-QUICKTREE   :use FAST algorithm for the alignment guide tree
571-TYPE=       :PROTEIN or DNA sequences
572-NEGATIVE    :protein alignment with negative values in matrix
573-OUTFILE=    :sequence alignment file name
574-OUTPUT=     :GCG, GDE, PHYLIP, PIR or NEXUS
575-OUTORDER=   :INPUT or ALIGNED
576-CASE        :LOWER or UPPER (for GDE output only)
577-SEQNOS=     :OFF or ON (for Clustal output only)
578-SEQNO_RANGE=:OFF or ON (NEW: for all output formats)
579-RANGE=m,n   :sequence range to write starting m to m+n.
580
581***Fast Pairwise Alignments:***
582-KTUPLE=n    :word size
583-TOPDIAGS=n  :number of best diags.
584-WINDOW=n    :window around best diags.
585-PAIRGAP=n   :gap penalty
586-SCORE       :PERCENT or ABSOLUTE
587
588
589***Slow Pairwise Alignments:***
590-PWMATRIX=    :Protein weight matrix=BLOSUM, PAM, GONNET, ID or filename
591-PWDNAMATRIX= :DNA weight matrix=IUB, CLUSTALW or filename
592-PWGAPOPEN=f  :gap opening penalty       
593-PWGAPEXT=f   :gap opening penalty
594
595
596***Multiple Alignments:***
597-NEWTREE=      :file for new guide tree
598-USETREE=      :file for old guide tree
599-MATRIX=       :Protein weight matrix=BLOSUM, PAM, GONNET, ID or filename
600-DNAMATRIX=    :DNA weight matrix=IUB, CLUSTALW or filename
601-GAPOPEN=f     :gap opening penalty       
602-GAPEXT=f      :gap extension penalty
603-ENDGAPS       :no end gap separation pen.
604-GAPDIST=n     :gap separation pen. range
605-NOPGAP        :residue-specific gaps off 
606-NOHGAP        :hydrophilic gaps off
607-HGAPRESIDUES= :list hydrophilic res.   
608-MAXDIV=n      :% ident. for delay
609-TYPE=         :PROTEIN or DNA
610-TRANSWEIGHT=f :transitions weighting
611
612
613***Profile Alignments:***
614-PROFILE      :Merge two alignments by profile alignment
615-NEWTREE1=    :file for new guide tree for profile1
616-NEWTREE2=    :file for new guide tree for profile2
617-USETREE1=    :file for old guide tree for profile1
618-USETREE2=    :file for old guide tree for profile2
619
620
621***Sequence to Profile Alignments:***
622-SEQUENCES   :Sequentially add profile2 sequences to profile1 alignment
623-NEWTREE=    :file for new guide tree
624-USETREE=    :file for old guide tree
625
626
627***Structure Alignments:***
628-NOSECSTR1     :do not use secondary structure-gap penalty mask for profile 1
629-NOSECSTR2     :do not use secondary structure-gap penalty mask for profile 2
630-SECSTROUT=STRUCTURE or MASK or BOTH or NONE   :output in alignment file
631-HELIXGAP=n    :gap penalty for helix core residues
632-STRANDGAP=n   :gap penalty for strand core residues
633-LOOPGAP=n     :gap penalty for loop regions
634-TERMINALGAP=n :gap penalty for structure termini
635-HELIXENDIN=n  :number of residues inside helix to be treated as terminal
636-HELIXENDOUT=n :number of residues outside helix to be treated as terminal
637-STRANDENDIN=n :number of residues inside strand to be treated as terminal
638-STRANDENDOUT=n:number of residues outside strand to be treated as terminal
639
640
641***Trees:***
642-OUTPUTTREE=nj OR phylip OR dist OR nexus
643-SEED=n        :seed number for bootstraps.
644-KIMURA        :use Kimura's correction.   
645-TOSSGAPS      :ignore positions with gaps.
646-BOOTLABELS=node OR branch :position of bootstrap values in tree display
647
648>>HELP 0 <<           Help for tree output format options
649
650Four output formats are offered: 1) Clustal, 2) Phylip, 3) Just the distances
6514) Nexus
652
653None of these formats displays the results graphically. Many packages can
654display trees in the the PHYLIP format 2) below. It can also be imported into
655the PHYLIP programs RETREE, DRAWTREE and DRAWGRAM for graphical display.
656NEXUS format trees can be read by PAUP and MacClade.
657
6581) Clustal format output.
659This format is verbose and lists all of the distances between the sequences and
660the number of alignment positions used for each. The tree is described at the
661end of the file. It lists the sequences that are joined at each alignment step
662and the branch lengths. After two sequences are joined, it is referred to later
663as a NODE. The number of a NODE is the number of the lowest sequence in that
664NODE.   
665
6662) Phylip format output.
667This format is the New Hampshire format, used by many phylogenetic analysis
668packages. It consists of a series of nested parentheses, describing the
669branching order, with the sequence names and branch lengths. It can be used by
670the RETREE, DRAWGRAM and DRAWTREE programs of the PHYLIP package to see the
671trees graphically. This is the same format used during multiple alignment for
672the guide trees.
673
674Use this format with NJplot (Manolo Gouy), supplied with Clustal W. Some other
675packages that can read and display New Hampshire format are TreeView (Mac/PC),
676TreeTool (UNIX), and Phylowin.
677
6783) The distances only.
679This format just outputs a matrix of all the pairwise distances in a format
680that can be used by the Phylip package. It used to be useful when one could not
681produce distances from protein sequences in the Phylip package but is now
682redundant (Protdist of Phylip 3.5 now does this).
683
6844) NEXUS FORMAT TREE. This format is used by several popular phylogeny programs,
685including PAUP and MacClade. The format is described fully in:
686Maddison, D. R., D. L. Swofford and W. P. Maddison.  1997.
687NEXUS: an extensible file format for systematic information.
688Systematic Biology 46:590-621.
689
6905) TOGGLE PHYLIP BOOTSTRAP POSITIONS
691By default, the bootstrap values are placed on the nodes of the phylip format
692output tree. This is inaccurate as the bootstrap values should be associated
693with the tree branches and not the nodes. However, this format can be read and
694displayed by TreeTool, TreeView and Phylowin. An option is available to
695correctly place the bootstrap values on the branches with which they are
696associated.
697
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