4 CLUSTAL 2.1 Multiple Sequence Alignments
9 >> HELP NEW << NEW FEATURES/OPTIONS
12 The UPGMA algorithm has been added to allow faster tree construction. The user now
13 has the choice of using Neighbour Joining or UPGMA. The default is still NJ, but the
14 user can change this by setting the clustering parameter.
16 -CLUSTERING= :NJ or UPGMA
20 A remove first iteration scheme has been added. This can be used to improve the final
21 alignment or improve the alignment at each stage of the progressive alignment. During the
22 iteration step each sequence is removed in turn and realigned. If the resulting alignment
23 is better than the previous alignment it is kept. This process is repeated until the score
24 converges (the score is not improved) or until the maximum number of iterations is
25 reached. The user can iterate at each step of the progressive alignment by setting the
26 iteration parameter to TREE or just on the final alignment by seting the iteration
27 parameter to ALIGNMENT. The default is no iteration. The maximum number of iterations can
28 be set using the numiter parameter. The default number of iterations is 3.
30 -ITERATION= :NONE or TREE or ALIGNMENT
32 -NUMITER=n :Maximum number of iterations to perform
36 -FULLHELP :Print out the complete help content
40 -MAXSEQLEN=n :Maximum allowed sequence length
42 -QUIET :Reduce console output to minimum
44 -STATS=file :Log some alignents statistics to file
47 >> HELP 1 << General help for CLUSTAL W (2.1)
49 Clustal W is a general purpose multiple alignment program for DNA or proteins.
51 SEQUENCE INPUT: all sequences must be in 1 file, one after another.
52 7 formats are automatically recognised: NBRF-PIR, EMBL-SWISSPROT,
53 Pearson (Fasta), Clustal (*.aln), GCG-MSF (Pileup), GCG9-RSF and GDE flat file.
54 All non-alphabetic characters (spaces, digits, punctuation marks) are ignored
55 except "-" which is used to indicate a GAP ("." in MSF-RSF).
57 To do a MULTIPLE ALIGNMENT on a set of sequences, use item 1 from this menu to
58 INPUT them; go to menu item 2 to do the multiple alignment.
60 PROFILE ALIGNMENTS (menu item 3) are used to align 2 alignments. Use this to
61 add a new sequence to an old alignment, or to use secondary structure to guide
62 the alignment process. GAPS in the old alignments are indicated using the "-"
63 character. PROFILES can be input in ANY of the allowed formats; just
64 use "-" (or "." for MSF-RSF) for each gap position.
66 PHYLOGENETIC TREES (menu item 4) can be calculated from old alignments (read in
67 with "-" characters to indicate gaps) OR after a multiple alignment while the
68 alignment is still in memory.
71 The program tries to automatically recognise the different file formats used
72 and to guess whether the sequences are amino acid or nucleotide. This is not
75 FASTA and NBRF-PIR formats are recognised by having a ">" as the first
76 character in the file.
78 EMBL-Swiss Prot formats are recognised by the letters
79 ID at the start of the file (the token for the entry name field).
81 CLUSTAL format is recognised by the word CLUSTAL at the beginning of the file.
83 GCG-MSF format is recognised by one of the following:
84 - the word PileUp at the start of the file.
85 - the word !!AA_MULTIPLE_ALIGNMENT or !!NA_MULTIPLE_ALIGNMENT
86 at the start of the file.
87 - the word MSF on the first line of the line, and the characters ..
88 at the end of this line.
90 GCG-RSF format is recognised by the word !!RICH_SEQUENCE at the beginning of
94 If 85% or more of the characters in the sequence are from A,C,G,T,U or N, the
95 sequence will be assumed to be nucleotide. This works in 97.3% of cases
99 >> HELP 2 << Help for multiple alignments
101 If you have already loaded sequences, use menu item 1 to do the complete
102 multiple alignment. You will be prompted for 2 output files: 1 for the
103 alignment itself; another to store a dendrogram that describes the similarity
104 of the sequences to each other.
106 Multiple alignments are carried out in 3 stages (automatically done from menu
107 item 1 ...Do complete multiple alignments now):
109 1) all sequences are compared to each other (pairwise alignments);
111 2) a dendrogram (like a phylogenetic tree) is constructed, describing the
112 approximate groupings of the sequences by similarity (stored in a file).
114 3) the final multiple alignment is carried out, using the dendrogram as a guide.
117 PAIRWISE ALIGNMENT parameters control the speed-sensitivity of the initial
120 MULTIPLE ALIGNMENT parameters control the gaps in the final multiple alignments.
123 RESET GAPS (menu item 7) will remove any new gaps introduced into the sequences
124 during multiple alignment if you wish to change the parameters and try again.
125 This only takes effect just before you do a second multiple alignment. You
126 can make phylogenetic trees after alignment whether or not this is ON.
127 If you turn this OFF, the new gaps are kept even if you do a second multiple
128 alignment. This allows you to iterate the alignment gradually. Sometimes, the
129 alignment is improved by a second or third pass.
131 SCREEN DISPLAY (menu item 8) can be used to send the output alignments to the
132 screen as well as to the output file.
134 You can skip the first stages (pairwise alignments; dendrogram) by using an
135 old dendrogram file (menu item 3); or you can just produce the dendrogram
136 with no final multiple alignment (menu item 2).
139 OUTPUT FORMAT: Menu item 9 (format options) allows you to choose from 6
140 different alignment formats (CLUSTAL, GCG, NBRF-PIR, PHYLIP, GDE, NEXUS, and FASTA).
144 >> HELP 3 << Help for pairwise alignment parameters
146 A distance is calculated between every pair of sequences and these are used to
147 construct the dendrogram which guides the final multiple alignment. The scores
148 are calculated from separate pairwise alignments. These can be calculated using
149 2 methods: dynamic programming (slow but accurate) or by the method of Wilbur
150 and Lipman (extremely fast but approximate).
152 You can choose between the 2 alignment methods using menu option 8. The
153 slow-accurate method is fine for short sequences but will be VERY SLOW for
154 many (e.g. >100) long (e.g. >1000 residue) sequences.
156 SLOW-ACCURATE alignment parameters:
157 These parameters do not have any affect on the speed of the alignments.
158 They are used to give initial alignments which are then rescored to give percent
159 identity scores. These % scores are the ones which are displayed on the
160 screen. The scores are converted to distances for the trees.
162 1) Gap Open Penalty: the penalty for opening a gap in the alignment.
163 2) Gap extension penalty: the penalty for extending a gap by 1 residue.
164 3) Protein weight matrix: the scoring table which describes the similarity
165 of each amino acid to each other.
166 4) DNA weight matrix: the scores assigned to matches and mismatches
167 (including IUB ambiguity codes).
170 FAST-APPROXIMATE alignment parameters:
172 These similarity scores are calculated from fast, approximate, global align-
173 ments, which are controlled by 4 parameters. 2 techniques are used to make
174 these alignments very fast: 1) only exactly matching fragments (k-tuples) are
175 considered; 2) only the 'best' diagonals (the ones with most k-tuple matches)
178 K-TUPLE SIZE: This is the size of exactly matching fragment that is used.
179 INCREASE for speed (max= 2 for proteins; 4 for DNA), DECREASE for sensitivity.
180 For longer sequences (e.g. >1000 residues) you may need to increase the default.
182 GAP PENALTY: This is a penalty for each gap in the fast alignments. It has
183 little affect on the speed or sensitivity except for extreme values.
185 TOP DIAGONALS: The number of k-tuple matches on each diagonal (in an imaginary
186 dot-matrix plot) is calculated. Only the best ones (with most matches) are
187 used in the alignment. This parameter specifies how many. Decrease for speed;
188 increase for sensitivity.
190 WINDOW SIZE: This is the number of diagonals around each of the 'best'
191 diagonals that will be used. Decrease for speed; increase for sensitivity.
194 >> HELP 4 << Help for multiple alignment parameters
196 These parameters control the final multiple alignment. This is the core of the
197 program and the details are complicated. To fully understand the use of the
198 parameters and the scoring system, you will have to refer to the documentation.
200 Each step in the final multiple alignment consists of aligning two alignments
201 or sequences. This is done progressively, following the branching order in
202 the GUIDE TREE. The basic parameters to control this are two gap penalties and
203 the scores for various identical-non-indentical residues.
205 1) and 2) The GAP PENALTIES are set by menu items 1 and 2. These control the
206 cost of opening up every new gap and the cost of every item in a gap.
207 Increasing the gap opening penalty will make gaps less frequent. Increasing
208 the gap extension penalty will make gaps shorter. Terminal gaps are not
211 3) The DELAY DIVERGENT SEQUENCES switch delays the alignment of the most
212 distantly related sequences until after the most closely related sequences have
213 been aligned. The setting shows the percent identity level required to delay
214 the addition of a sequence; sequences that are less identical than this level
215 to any other sequences will be aligned later.
219 4) The TRANSITION WEIGHT gives transitions (A <--> G or C <--> T
220 i.e. purine-purine or pyrimidine-pyrimidine substitutions) a weight between 0
221 and 1; a weight of zero means that the transitions are scored as mismatches,
222 while a weight of 1 gives the transitions the match score. For distantly related
223 DNA sequences, the weight should be near to zero; for closely related sequences
224 it can be useful to assign a higher score.
227 5) PROTEIN WEIGHT MATRIX leads to a new menu where you are offered a choice of
228 weight matrices. The default for proteins in version 1.8 is the PAM series
229 derived by Gonnet and colleagues. Note, a series is used! The actual matrix
230 that is used depends on how similar the sequences to be aligned at this
231 alignment step are. Different matrices work differently at each evolutionary
234 6) DNA WEIGHT MATRIX leads to a new menu where a single matrix (not a series)
235 can be selected. The default is the matrix used by BESTFIT for comparison of
236 nucleic acid sequences.
238 Further help is offered in the weight matrix menu.
241 7) In the weight matrices, you can use negative as well as positive values if
242 you wish, although the matrix will be automatically adjusted to all positive
243 scores, unless the NEGATIVE MATRIX option is selected.
245 8) PROTEIN GAP PARAMETERS displays a menu allowing you to set some Gap Penalty
246 options which are only used in protein alignments.
249 >> HELP A << Help for protein gap parameters.
251 1) RESIDUE SPECIFIC PENALTIES are amino acid specific gap penalties that reduce
252 or increase the gap opening penalties at each position in the alignment or
253 sequence. See the documentation for details. As an example, positions that
254 are rich in glycine are more likely to have an adjacent gap than positions that
257 2) 3) HYDROPHILIC GAP PENALTIES are used to increase the chances of a gap within
258 a run (5 or more residues) of hydrophilic amino acids; these are likely to
259 be loop or random coil regions where gaps are more common. The residues that
260 are "considered" to be hydrophilic are set by menu item 3.
262 4) GAP SEPARATION DISTANCE tries to decrease the chances of gaps being too
263 close to each other. Gaps that are less than this distance apart are penalised
264 more than other gaps. This does not prevent close gaps; it makes them less
265 frequent, promoting a block-like appearance of the alignment.
267 5) END GAP SEPARATION treats end gaps just like internal gaps for the purposes
268 of avoiding gaps that are too close (set by GAP SEPARATION DISTANCE above).
269 If you turn this off, end gaps will be ignored for this purpose. This is
270 useful when you wish to align fragments where the end gaps are not biologically
274 >> HELP 5 << Help for output format options.
276 Several output formats are offered. You can choose any (or all 6 if you wish).
278 CLUSTAL format output is a self explanatory alignment format. It shows the
279 sequences aligned in blocks. It can be read in again at a later date to
280 (for example) calculate a phylogenetic tree or add a new sequence with a
283 GCG output can be used by any of the GCG programs that can work on multiple
284 alignments (e.g. PRETTY, PROFILEMAKE, PLOTALIGN). It is the same as the GCG
285 .msf format files (multiple sequence file); new in version 7 of GCG.
287 Fasta output cis widely used because of it's simplicity. Each sequence name is
288 preceeded by a '>'-sign. The sequence itself is printed out in the following lines
290 PHYLIP format output can be used for input to the PHYLIP package of Joe
291 Felsenstein. This is an extremely widely used package for doing every
292 imaginable form of phylogenetic analysis (MUCH more than the the modest intro-
293 duction offered by this program).
295 NBRF-PIR: this is the same as the standard PIR format with ONE ADDITION. Gap
296 characters "-" are used to indicate the positions of gaps in the multiple
297 alignment. These files can be re-used as input in any part of clustal that
298 allows sequences (or alignments or profiles) to be read in.
300 GDE: this is the flat file format used by the GDE package of Steven Smith.
302 NEXUS: the format used by several phylogeny programs, including PAUP and
305 GDE OUTPUT CASE: sequences in GDE format may be written in either upper or
308 CLUSTALW SEQUENCE NUMBERS: residue numbers may be added to the end of the
309 alignment lines in clustalw format.
311 OUTPUT ORDER is used to control the order of the sequences in the output
312 alignments. By default, the order corresponds to the order in which the
313 sequences were aligned (from the guide tree-dendrogram), thus automatically
314 grouping closely related sequences. This switch can be used to set the order
315 to the same as the input file.
317 PARAMETER OUTPUT: This option allows you to save all your parameter settings
318 in a parameter file. This file can be used subsequently to rerun Clustal W
319 using the same parameters.
322 >> HELP 6 << Help for profile and structure alignments
324 By PROFILE ALIGNMENT, we mean alignment using existing alignments. Profile
325 alignments allow you to store alignments of your favourite sequences and add
326 new sequences to them in small bunches at a time. A profile is simply an
327 alignment of one or more sequences (e.g. an alignment output file from CLUSTAL
328 W). Each input can be a single sequence. One or both sets of input sequences
329 may include secondary structure assignments or gap penalty masks to guide the
332 The profiles can be in any of the allowed input formats with "-" characters
333 used to specify gaps (except for MSF-RSF where "." is used).
335 You have to specify the 2 profiles by choosing menu items 1 and 2 and giving
336 2 file names. Then Menu item 3 will align the 2 profiles to each other.
337 Secondary structure masks in either profile can be used to guide the alignment.
339 Menu item 4 will take the sequences in the second profile and align them to
340 the first profile, 1 at a time. This is useful to add some new sequences to
341 an existing alignment, or to align a set of sequences to a known structure.
342 In this case, the second profile would not be pre-aligned.
345 The alignment parameters can be set using menu items 5, 6 and 7. These are
346 EXACTLY the same parameters as used by the general, automatic multiple
347 alignment procedure. The general multiple alignment procedure is simply a
348 series of profile alignments. Carrying out a series of profile alignments on
349 larger and larger groups of sequences, allows you to manually build up a
350 complete alignment, if necessary editing intermediate alignments.
352 SECONDARY STRUCTURE OPTIONS. Menu Option 0 allows you to set 2D structure
353 parameters. If a solved structure is available, it can be used to guide the
354 alignment by raising gap penalties within secondary structure elements, so
355 that gaps will preferentially be inserted into unstructured surface loops.
356 Alternatively, a user-specified gap penalty mask can be supplied directly.
358 A gap penalty mask is a series of numbers between 1 and 9, one per position in
359 the alignment. Each number specifies how much the gap opening penalty is to be
360 raised at that position (raised by multiplying the basic gap opening penalty
361 by the number) i.e. a mask figure of 1 at a position means no change
362 in gap opening penalty; a figure of 4 means that the gap opening penalty is
363 four times greater at that position, making gaps 4 times harder to open.
365 The format for gap penalty masks and secondary structure masks is explained
366 in the help under option 0 (secondary structure options).
369 >> HELP B << Help for secondary structure - gap penalty masks
371 The use of secondary structure-based penalties has been shown to improve the
372 accuracy of multiple alignment. Therefore CLUSTAL W now allows gap penalty
373 masks to be supplied with the input sequences. The masks work by raising gap
374 penalties in specified regions (typically secondary structure elements) so that
375 gaps are preferentially opened in the less well conserved regions (typically
378 Options 1 and 2 control whether the input secondary structure information or
379 gap penalty masks will be used.
381 Option 3 controls whether the secondary structure and gap penalty masks should
382 be included in the output alignment.
384 Options 4 and 5 provide the value for raising the gap penalty at core Alpha
385 Helical (A) and Beta Strand (B) residues. In CLUSTAL format, capital residues
386 denote the A and B core structure notation. The basic gap penalties are
387 multiplied by the amount specified.
389 Option 6 provides the value for the gap penalty in Loops. By default this
390 penalty is not raised. In CLUSTAL format, loops are specified by "." in the
391 secondary structure notation.
393 Option 7 provides the value for setting the gap penalty at the ends of
394 secondary structures. Ends of secondary structures are observed to grow
395 and-or shrink in related structures. Therefore by default these are given
396 intermediate values, lower than the core penalties. All secondary structure
397 read in as lower case in CLUSTAL format gets the reduced terminal penalty.
399 Options 8 and 9 specify the range of structure termini for the intermediate
400 penalties. In the alignment output, these are indicated as lower case.
401 For Alpha Helices, by default, the range spans the end helical turn. For
402 Beta Strands, the default range spans the end residue and the adjacent loop
403 residue, since sequence conservation often extends beyond the actual H-bonded
406 CLUSTAL W can read the masks from SWISS-PROT, CLUSTAL or GDE format input
407 files. For many 3-D protein structures, secondary structure information is
408 recorded in the feature tables of SWISS-PROT database entries. You should
409 always check that the assignments are correct - some are quite inaccurate.
410 CLUSTAL W looks for SWISS-PROT HELIX and STRAND assignments e.g.
415 The structure and penalty masks can also be read from CLUSTAL alignment format
416 as comment lines beginning "!SS_" or "!GM_" e.g.
418 !SS_HBA_HUMA ..aaaAAAAAAAAAAaaa.aaaAAAAAAAAAAaaaaaaAaaa.........aaaAAAAAA
419 !GM_HBA_HUMA 112224444444444222122244444444442222224222111111111222444444
420 HBA_HUMA VLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLSHGSAQVKGHGK
422 Note that the mask itself is a set of numbers between 1 and 9 each of which is
423 assigned to the residue(s) in the same column below.
425 In GDE flat file format, the masks are specified as text and the names must
426 begin with "SS_ or "GM_.
428 Either a structure or penalty mask or both may be used. If both are included in
429 an alignment, the user will be asked which is to be used.
432 >> HELP C << Help for secondary structure - gap penalty mask output options
434 The options in this menu let you choose whether or not to include the masks
435 in the CLUSTAL W output alignments. Showing both is useful for understanding
436 how the masks work. The secondary structure information is itself very useful
437 in judging the alignment quality and in seeing how residue conservation
438 patterns vary with secondary structure.
441 >> HELP 7 << Help for phylogenetic trees
443 1) Before calculating a tree, you must have an ALIGNMENT in memory. This can be
444 input in any format or you should have just carried out a full multiple
445 alignment and the alignment is still in memory.
448 *************** Remember YOU MUST ALIGN THE SEQUENCES FIRST!!!! ***************
451 The methods used are NJ (Neighbour Joining) and UPGMA. First
452 you calculate distances (percent divergence) between all pairs of sequence from
453 a multiple alignment; second you apply the NJ or UPGMA method to the distance matrix.
455 2) EXCLUDE POSITIONS WITH GAPS? With this option, any alignment positions where
456 ANY of the sequences have a gap will be ignored. This means that 'like' will be
457 compared to 'like' in all distances, which is highly desirable. It also
458 automatically throws away the most ambiguous parts of the alignment, which are
459 concentrated around gaps (usually). The disadvantage is that you may throw away
460 much of the data if there are many gaps (which is why it is difficult for us to
461 make it the default).
465 3) CORRECT FOR MULTIPLE SUBSTITUTIONS? For small divergence (say <10%) this
466 option makes no difference. For greater divergence, it corrects for the fact
467 that observed distances underestimate actual evolutionary distances. This is
468 because, as sequences diverge, more than one substitution will happen at many
469 sites. However, you only see one difference when you look at the present day
470 sequences. Therefore, this option has the effect of stretching branch lengths
471 in trees (especially long branches). The corrections used here (for DNA or
472 proteins) are both due to Motoo Kimura. See the documentation for details.
474 Where possible, this option should be used. However, for VERY divergent
475 sequences, the distances cannot be reliably corrected. You will be warned if
476 this happens. Even if none of the distances in a data set exceed the reliable
477 threshold, if you bootstrap the data, some of the bootstrap distances may
478 randomly exceed the safe limit.
480 4) To calculate a tree, use option 4 (DRAW TREE NOW). This gives an UNROOTED
481 tree and all branch lengths. The root of the tree can only be inferred by
482 using an outgroup (a sequence that you are certain branches at the outside
483 of the tree .... certain on biological grounds) OR if you assume a degree
484 of constancy in the 'molecular clock', you can place the root in the 'middle'
485 of the tree (roughly equidistant from all tips).
487 5) TOGGLE PHYLIP BOOTSTRAP POSITIONS
488 By default, the bootstrap values are correctly placed on the tree branches of
489 the phylip format output tree. The toggle allows them to be placed on the
490 nodes, which is incorrect, but some display packages (e.g. TreeTool, TreeView
491 and Phylowin) only support node labelling but not branch labelling. Care
492 should be taken to note which branches and labels go together.
494 6) OUTPUT FORMATS: four different formats are allowed. None of these displays
495 the tree visually. Useful display programs accepting PHYLIP format include
496 NJplot (from Manolo Gouy and supplied with Clustal W), TreeView (Mac-PC), and
497 PHYLIP itself - OR get the PHYLIP package and use the tree drawing facilities
498 there. (Get the PHYLIP package anyway if you are interested in trees). The
499 NEXUS format can be read into PAUP or MacClade.
502 >> HELP 8 << Help for choosing a weight matrix
504 For protein alignments, you use a weight matrix to determine the similarity of
505 non-identical amino acids. For example, Tyr aligned with Phe is usually judged
506 to be 'better' than Tyr aligned with Pro.
508 There are three 'in-built' series of weight matrices offered. Each consists of
509 several matrices which work differently at different evolutionary distances. To
510 see the exact details, read the documentation. Crudely, we store several
511 matrices in memory, spanning the full range of amino acid distance (from almost
512 identical sequences to highly divergent ones). For very similar sequences, it
513 is best to use a strict weight matrix which only gives a high score to
514 identities and the most favoured conservative substitutions. For more divergent
515 sequences, it is appropriate to use "softer" matrices which give a high score
516 to many other frequent substitutions.
518 1) BLOSUM (Henikoff). These matrices appear to be the best available for
519 carrying out database similarity (homology searches). The matrices used are:
520 Blosum 80, 62, 45 and 30. (BLOSUM was the default in earlier Clustal W
523 2) PAM (Dayhoff). These have been extremely widely used since the late '70s.
524 We use the PAM 20, 60, 120 and 350 matrices.
526 3) GONNET. These matrices were derived using almost the same procedure as the
527 Dayhoff one (above) but are much more up to date and are based on a far larger
528 data set. They appear to be more sensitive than the Dayhoff series. We use the
529 GONNET 80, 120, 160, 250 and 350 matrices. This series is the default for
530 Clustal W version 1.8.
532 We also supply an identity matrix which gives a score of 1.0 to two identical
533 amino acids and a score of zero otherwise. This matrix is not very useful.
534 Alternatively, you can read in your own (just one matrix, not a series).
536 A new matrix can be read from a file on disk, if the filename consists only
537 of lower case characters. The values in the new weight matrix must be integers
538 and the scores should be similarities. You can use negative as well as positive
539 values if you wish, although the matrix will be automatically adjusted to all
544 For DNA, a single matrix (not a series) is used. Two hard-coded matrices are
548 1) IUB. This is the default scoring matrix used by BESTFIT for the comparison
549 of nucleic acid sequences. X's and N's are treated as matches to any IUB
550 ambiguity symbol. All matches score 1.9; all mismatches for IUB symbols score 0.
553 2) CLUSTALW(1.6). The previous system used by Clustal W, in which matches score
554 1.0 and mismatches score 0. All matches for IUB symbols also score 0.
556 INPUT FORMAT The format used for a new matrix is the same as the BLAST program.
557 Any lines beginning with a # character are assumed to be comments. The first
558 non-comment line should contain a list of amino acids in any order, using the
559 1 letter code, followed by a * character. This should be followed by a square
560 matrix of integer scores, with one row and one column for each amino acid. The
561 last row and column of the matrix (corresponding to the * character) contain
562 the minimum score over the whole matrix.
565 >> HELP 9 << Help for command line parameters
569 -INFILE=file.ext :input sequences.
570 -PROFILE1=file.ext and -PROFILE2=file.ext :profiles (old alignment).
575 -OPTIONS :list the command line parameters
576 -HELP or -CHECK :outline the command line params.
577 -FULLHELP :output full help content.
578 -ALIGN :do full multiple alignment.
579 -TREE :calculate NJ tree.
580 -PIM :output percent identity matrix (while calculating the tree)
581 -BOOTSTRAP(=n) :bootstrap a NJ tree (n= number of bootstraps; def. = 1000).
582 -CONVERT :output the input sequences in a different file format.
585 PARAMETERS (set things)
587 ***General settings:****
588 -INTERACTIVE :read command line, then enter normal interactive menus
589 -QUICKTREE :use FAST algorithm for the alignment guide tree
590 -TYPE= :PROTEIN or DNA sequences
591 -NEGATIVE :protein alignment with negative values in matrix
592 -OUTFILE= :sequence alignment file name
593 -OUTPUT= :CLUSTAL(default), GCG, GDE, PHYLIP, PIR, NEXUS and FASTA
594 -OUTORDER= :INPUT or ALIGNED
595 -CASE :LOWER or UPPER (for GDE output only)
596 -SEQNOS= :OFF or ON (for Clustal output only)
597 -SEQNO_RANGE=:OFF or ON (NEW: for all output formats)
598 -RANGE=m,n :sequence range to write starting m to m+n
599 -MAXSEQLEN=n :maximum allowed input sequence length
600 -QUIET :Reduce console output to minimum
601 -STATS= :Log some alignents statistics to file
603 ***Fast Pairwise Alignments:***
605 -TOPDIAGS=n :number of best diags.
606 -WINDOW=n :window around best diags.
607 -PAIRGAP=n :gap penalty
608 -SCORE :PERCENT or ABSOLUTE
611 ***Slow Pairwise Alignments:***
612 -PWMATRIX= :Protein weight matrix=BLOSUM, PAM, GONNET, ID or filename
613 -PWDNAMATRIX= :DNA weight matrix=IUB, CLUSTALW or filename
614 -PWGAPOPEN=f :gap opening penalty
615 -PWGAPEXT=f :gap opening penalty
618 ***Multiple Alignments:***
619 -NEWTREE= :file for new guide tree
620 -USETREE= :file for old guide tree
621 -MATRIX= :Protein weight matrix=BLOSUM, PAM, GONNET, ID or filename
622 -DNAMATRIX= :DNA weight matrix=IUB, CLUSTALW or filename
623 -GAPOPEN=f :gap opening penalty
624 -GAPEXT=f :gap extension penalty
625 -ENDGAPS :no end gap separation pen.
626 -GAPDIST=n :gap separation pen. range
627 -NOPGAP :residue-specific gaps off
628 -NOHGAP :hydrophilic gaps off
629 -HGAPRESIDUES= :list hydrophilic res.
630 -MAXDIV=n :% ident. for delay
631 -TYPE= :PROTEIN or DNA
632 -TRANSWEIGHT=f :transitions weighting
633 -ITERATION= :NONE or TREE or ALIGNMENT
634 -NUMITER=n :maximum number of iterations to perform
635 -NOWEIGHTS :disable sequence weighting
638 ***Profile Alignments:***
639 -PROFILE :Merge two alignments by profile alignment
640 -NEWTREE1= :file for new guide tree for profile1
641 -NEWTREE2= :file for new guide tree for profile2
642 -USETREE1= :file for old guide tree for profile1
643 -USETREE2= :file for old guide tree for profile2
646 ***Sequence to Profile Alignments:***
647 -SEQUENCES :Sequentially add profile2 sequences to profile1 alignment
648 -NEWTREE= :file for new guide tree
649 -USETREE= :file for old guide tree
652 ***Structure Alignments:***
653 -NOSECSTR1 :do not use secondary structure-gap penalty mask for profile 1
654 -NOSECSTR2 :do not use secondary structure-gap penalty mask for profile 2
655 -SECSTROUT=STRUCTURE or MASK or BOTH or NONE :output in alignment file
656 -HELIXGAP=n :gap penalty for helix core residues
657 -STRANDGAP=n :gap penalty for strand core residues
658 -LOOPGAP=n :gap penalty for loop regions
659 -TERMINALGAP=n :gap penalty for structure termini
660 -HELIXENDIN=n :number of residues inside helix to be treated as terminal
661 -HELIXENDOUT=n :number of residues outside helix to be treated as terminal
662 -STRANDENDIN=n :number of residues inside strand to be treated as terminal
663 -STRANDENDOUT=n:number of residues outside strand to be treated as terminal
667 -OUTPUTTREE=nj OR phylip OR dist OR nexus
668 -SEED=n :seed number for bootstraps.
669 -KIMURA :use Kimura's correction.
670 -TOSSGAPS :ignore positions with gaps.
671 -BOOTLABELS=node OR branch :position of bootstrap values in tree display
672 -CLUSTERING= :NJ or UPGMA
675 >> HELP 0 << Help for tree output format options
677 Four output formats are offered: 1) Clustal, 2) Phylip, 3) Just the distances
680 None of these formats displays the results graphically. Many packages can
681 display trees in the the PHYLIP format 2) below. It can also be imported into
682 the PHYLIP programs RETREE, DRAWTREE and DRAWGRAM for graphical display.
683 NEXUS format trees can be read by PAUP and MacClade.
685 1) Clustal format output.
686 This format is verbose and lists all of the distances between the sequences and
687 the number of alignment positions used for each. The tree is described at the
688 end of the file. It lists the sequences that are joined at each alignment step
689 and the branch lengths. After two sequences are joined, it is referred to later
690 as a NODE. The number of a NODE is the number of the lowest sequence in that
693 2) Phylip format output.
694 This format is the New Hampshire format, used by many phylogenetic analysis
695 packages. It consists of a series of nested parentheses, describing the
696 branching order, with the sequence names and branch lengths. It can be used by
697 the RETREE, DRAWGRAM and DRAWTREE programs of the PHYLIP package to see the
698 trees graphically. This is the same format used during multiple alignment for
701 Use this format with NJplot (Manolo Gouy), supplied with Clustal W. Some other
702 packages that can read and display New Hampshire format are TreeView (Mac/PC),
703 TreeTool (UNIX), and Phylowin.
705 3) The distances only.
706 This format just outputs a matrix of all the pairwise distances in a format
707 that can be used by the Phylip package. It used to be useful when one could not
708 produce distances from protein sequences in the Phylip package but is now
709 redundant (Protdist of Phylip 3.5 now does this).
711 4) NEXUS FORMAT TREE. This format is used by several popular phylogeny programs,
712 including PAUP and MacClade. The format is described fully in:
713 Maddison, D. R., D. L. Swofford and W. P. Maddison. 1997.
714 NEXUS: an extensible file format for systematic information.
715 Systematic Biology 46:590-621.
717 5) TOGGLE PHYLIP BOOTSTRAP POSITIONS
718 By default, the bootstrap values are placed on the nodes of the phylip format
719 output tree. This is inaccurate as the bootstrap values should be associated
720 with the tree branches and not the nodes. However, this format can be read and
721 displayed by TreeTool, TreeView and Phylowin. An option is available to
722 correctly place the bootstrap values on the branches with which they are