+ 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 X: ++ ++
++++Jeannmougin,F., Thompson,J.D., Gouy,M., Higgins,D.G. and Gibson,T.J. (1998) ++Multiple sequence alignment with Clustal X. Trends Biochem Sci, 23, 403-5. ++
++++ ++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: ++ ++
++++http://www-igbmc.u-strasbg.fr/BioInfo/ ++
++++
++ Back to Index ++ ++++You can get the latest version of the ClustalX program here: ++
++ ++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. ++++
++
++++Clustal X is a windows interface for the ClustalW multiple sequence alignment ++program. It provides an integrated environment for performing multiple sequence ++and profile alignments and analysing the results. The sequence alignment is ++displayed in a window on the screen. A versatile coloring scheme has been ++incorporated allowing you to highlight conserved features in the alignment. ++The pull-down menus at the top of the window allow you to select all the ++options required for traditional multiple sequence and profile alignment. ++
++++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.) ++
++++
++++
++Sequences and profiles (a term for pre-existing alignments) are input using ++the FILE menu. Invalid options will be disabled. All sequences must be included ++into 1 file. 7 formats are automatically recognised: NBRF/PIR, EMBL/SWISSPROT, ++Pearson (Fasta), Clustal (*.aln), GCG/MSF (Pileup), GCG9 RSF and GDE flat file. ++All non-alphabetic characters (spaces, digits, punctuation marks) are ignored ++except "-" which is used to indicate a GAP ("." in MSF/RSF). ++
++++
++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 (termed profiles). Profiles are also used to add a new ++sequence to an old alignment, or to use secondary structure to guide the ++alignment process. GAPS in the old alignments are indicated using the "-" ++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 can be calculated from old alignments (read in with "-" ++characters to indicate gaps) OR after a multiple alignment while the alignment ++is still displayed. ++
++++
++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). ++
++++A line above the alignment 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 ++++ ++
++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. ++
++++
++++
++ Back to Index ++++
++++LOAD SEQUENCES reads sequences from one of 7 file formats, replacing any ++sequences that are already loaded. All sequences must be in 1 file. The formats ++that are automatically recognised are: NBRF/PIR, EMBL/SWISSPROT, Pearson ++(Fasta), Clustal (*.aln), GCG/MSF (Pileup), GCG9/RSF and GDE flat file. All ++non-alphabetic characters (spaces, digits, punctuation marks) are ignored ++except "-" 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: ++
++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 six output formats: ++CLUSTAL, NBRF/PIR, GCG/MSF, PHYLIP, NEXUS or GDE. All sequences are written ++to a single file. Options are available to save a range of the alignment, ++switch between UPPER/LOWER case for GDE files, and to output SEQUENCE NUMBERING ++for CLUSTAL files. ++
++++LOAD PROFILE 1 reads sequences in the same 7 file formats, replacing any ++sequences already loaded as Profile 1. This option will also remove any ++sequences which are loaded in Profile 2. ++
++++LOAD PROFILE 2 reads sequences in the same 7 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 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. ++
++++
++++
++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, A3 or US Letter size ++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. ++
++++
++ Back to Index ++++
++++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. ++
++++To search for a string of residues in the sequences, select the sequences to be ++searched by clicking on the sequence names. You can then enter the string to ++search for by selecting the SEARCH FOR STRING option. If the string is found in ++any of the sequences selected, the sequence name and column number is printed ++below the sequence display. ++
++++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 divergent ++sequences from an alignment, or if an alignment has been realigned. ++
++++
++ Back to Index ++++
++++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 stages (pairwise alignments; guide tree) by using an old ++guide tree file (DO ALIGNMENT FROM GUIDE TREE); or you can just produce the ++guide tree with no final multiple alignment (PRODUCE GUIDE TREE ONLY). ++
++++
++++REALIGN SELECTED SEQUENCES is used to realign badly aligned sequences in the ++alignment. Sequences can be selected by clicking on the sequence names - see ++Editing Alignments for more details. The unselected sequences are then 'fixed' ++and a profile is made including only the unselected sequences. Each of the ++selected sequences in turn is then realigned to this profile. The realigned ++sequences will be displayed as a group at the end the alignment. ++
++++
++++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. ++
++++(SECONDARY STRUCTURE PARAMETERS, for use with the Profile Alignment Mode only, ++allows you to set various parameters only used with gap penalty masks.) ++
++++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. ++
++++
++++
++You can choose from 6 different alignment formats (CLUSTAL, GCG, NBRF/PIR, ++PHYLIP, GDE and NEXUS). You can choose more than one (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 in new sequences by 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. ++
++++NEXUS format is used by several phylogeny programs, including PAUP and ++MacClade. ++
++++PHYLIP format output can be used for input to the PHYLIP package of Joe ++Felsenstein. This is a very widely used package for doing every imaginable ++form of phylogenetic analysis (MUCH more than the the modest introduction ++offered by this program). ++
++++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 and is understood ++by SEQLAB in GCG 9 or later. ++
++++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 uses the order in which the sequences were aligned ++(from the guide tree/dendrogram), thus automatically grouping closely related ++sequences. It can be switched to be the same as the original input order. ++
++++PARAMETER OUTPUT: This option will save all your parameter settings in a ++parameter file (suffix .par) during alignment. The file can be subsequently ++used to rerun ClustalW using the same 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 fast enough for short sequences but will be ++VERY SLOW for many (e.g. >100) 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 you 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 you to read in a comparison table from a file. ++
++++See the Multiple alignment parameters, MATRIX option below for details of the ++matrix input format. ++
++++
++++ ++FAST-APPROXIMATE alignment parameters: ++ ++
++++These similarity scores are calculated from fast, approximate, global align- ++ments, which are controlled by 4 parameters. 2 techniques are used to make ++these alignments very fast: 1) only exactly matching fragments (k-tuples) are ++considered; 2) only the 'best' diagonals (the ones with most k-tuple matches) ++are used. ++
++++GAP PENALTY: This is a penalty for each gap in the fast alignments. It has ++little effect on the speed or sensitivity except for extreme values. ++
++++K-TUPLE SIZE: This is the size of exactly matching fragment that is used. ++INCREASE for speed (max= 2 for proteins; 4 for DNA), DECREASE for sensitivity. ++For longer sequences (e.g. >1000 residues) you may wish to increase the ++default. ++
++++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 default is set to 0.5. ++
++++
++++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 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 currently ++used are: Blosum 80, 62, 45 and 30. BLOSUM was the default in earlier Clustal X ++versions. ++
++++2) PAM (Dayhoff). These have been extremely widely used since the late '70s. We ++currently use the PAM 20, 60, 120, 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 ++currently use the GONNET 80, 120, 160, 250 and 350 matrices. This series is the ++default for Clustal X version 1.8. ++
++++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 you to read in a comparison matrix from a file. ++This can be either a single matrix or a series of matrices (see below for ++format). ++
++++
++++DNA WEIGHT MATRIX option allows you to select a single matrix (not a series) ++used for aligning nucleic acid sequences. Two hard-coded matrices are available: ++
++++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). A 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 you to read in a nucleic acid 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 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. ++
++++
++++
++ Back to Index ++++
++++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 ++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 old ++guide tree files (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 set 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. ++
++++ ++SECONDARY STRUCTURE PARAMETERS ++ ++
++++Use this menu to set secondary structure options. If a solved structure is ++known, it can be used to guide the alignment by raising gap penalties within ++secondary structure elements, so that gaps will preferentially be inserted into ++unstructured surface loop regions. Alternatively, a user-specified gap penalty ++mask can be supplied for a similar purpose. ++
++++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 help section. ++
++++
++ Back to Index ++++
++++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 2D-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 2D-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 ++known to grow or shrink, comparing related structures. Therefore by default ++these are given intermediate values, lower than the core penalties. All ++secondary structure read in as lower case in CLUSTAL format gets the reduced ++terminal penalty. ++
++++The HELIX and STRAND TERMINAL POSITIONS options specify the range of structure ++termini for the intermediate penalties. In the alignment output, these are ++indicated as lower case. For Alpha Helices, by default, the range spans the ++end-helical turn (3 residues). For Beta Strands, the default range spans the ++end residue and the adjacent loop residue, since sequence conservation often ++extends beyond the actual H-bonded Beta Strand. ++
++++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 ++++ ++
++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. ++
++++
++++
++ Back to Index ++++
++++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 large number of bootstrap replicates (1000 is ++recommended, even if it means running the program for an hour on a slow ++computer). You can also supply a seed number for the random number generator ++here. Different runs with the same seed will give the same answer. See the ++documentation for more details. ++
++++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). ++
++++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 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. ++
++++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. ++
++++
++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 TREE. This format is verbose and lists all of the distances ++between the sequences and the number of alignment positions used for each. The ++tree is described at the end of the file. It lists the sequences that are ++joined at each alignment step and the branch lengths. After two sequences are ++joined, it is referred to later as a NODE. The number of a NODE is the number ++of the lowest sequence in that NODE. ++
++++2) PHYLIP FORMAT TREE. This format is the New Hampshire format, used by many ++phylogenetic analysis packages. It consists of a series of nested parentheses, ++describing the branching order, with the sequence names and branch lengths. It ++can be read by the NJPLOT program distributed with ClustalX. It can also be ++used by the RETREE, DRAWGRAM and DRAWTREE programs of the PHYLIP package to see ++the trees graphically. This is the same format used during multiple alignment ++for the guide trees. Some other packages that can read and display New ++Hampshire format are TreeTool, TreeView, and Phylowin. ++
++++3) PHYLIP DISTANCE MATRIX. This format just outputs a matrix of all the ++pairwise distances in a format that can be used by the PHYLIP package. It used ++to be useful when one could not produce distances from protein sequences in the ++Phylip package but is now redundant (PROTDIST of Phylip 3.5 now does this). ++
++++4) NEXUS FORMAT TREE. This format is used by several popular phylogeny programs, ++including PAUP and MacClade. The format is described fully in: ++Maddison, D. R., D. L. Swofford and W. P. Maddison. 1997. ++NEXUS: an extensible file format for systematic information. ++Systematic Biology 46:590-621. ++
++++BOOTSTRAP LABELS ON: By default, the bootstrap values are correctly placed on ++the tree branches of the phylip format output tree. The toggle allows them to ++be placed on the nodes, which is incorrect, but some display packages (e.g. ++TreeTool, TreeView and Phylowin) only support node labelling but not branch ++labelling. Care should be taken to note which branches and labels go together. ++
++++
++++
++ Back to Index ++++
++++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 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 BACKGROUND COLORING option shows the sequence residues using a ++black character on a colored background. It can be switched off to show ++residues as a colored character on a white background. ++
++++Either BLACK AND WHITE or DEFAULT COLOR options can be selected. The Color ++option looks first for the color parameter file (as described above) and, if no ++file is found, uses the default residue-specific colors. ++
++++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. ++
++++
++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 ++
++++@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. ++ 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 ++++
++++
++Clustal X provides an indication of the quality of an alignment by plotting ++a 'conservation score' for each column of the alignment. A high score indicates ++a well-conserved column; a low score indicates low conservation. The quality ++curve is drawn below the alignment. ++
++++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. ++
++++
++++
++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. ++
++++
++++
++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. ++
++++
++++PROTEIN WEIGHT MATRIX: the scoring table which describes the similarity of each ++amino acid to each other. The matrix is used to calculate the sequence- ++weighted profile scores. There are four 'in-built' Log-Odds matrices offered: ++the Gonnet PAM 80, 120, 250, 350 matrices. A more stringent matrix which only ++gives a high score to identities and the most favoured conservative ++substitutions, may be more suitable when the sequences are closely related. For ++more divergent sequences, it is appropriate to use "softer" matrices which give ++a high score to many other frequent substitutions. This option automatically ++recalculates the low-scoring segments. ++
++++
++++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 1.0; all mismatches for IUB symbols score ++0.9. ++
++++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. ++
++++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. ++
++++
++You can customise the column 'quality scores' plotted underneath the alignment ++display using the following options. ++
++++SCORE PLOT SCALE: this is a scalar value from 1 to 10, which can be used to ++change the scale of the quality score plot. ++
++++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. ++
++++For details of the quality score calculations, see the CALCULATION section ++below. ++
++++
++++ ++SHOW LOW-SCORING SEGMENTS ++ ++
++++The low-scoring segment display can be toggled on or off. This option does not ++recalculate the profile scores. ++
++++
++++ ++SHOW EXCEPTIONAL RESIDUES ++ ++
++++This option highlights individual residues which score badly in the alignment ++quality calculations. Residues which score exceptionally low are highlighted by ++using a white character on a grey background. ++
++++ ++SAVE QUALITY SCORES TO FILE ++ ++
++++The quality scores that are plotted underneath the alignment display can also ++be saved in a text file. Each column in the alignment is written on one line in ++the output file, with the value of the quality score at the end of the line. ++Only the sequences currently selected in the display are written to the file. ++One use for quality scores is to color residues in a protein structure by ++sequence conservation. In this way conserved surface residues can be ++highlighted to locate functional regions such as ligand-binding sites. ++
++++
++++
++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. ++
++++
++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.) ++
++++
++++
++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 ++| Parameter | ++Description | ++
| -PROFILE1=file.ext and -PROFILE2=file.ext | ++profiles (aligned sequences) | ++
| Parameter | ++Description | ++
| -HELP or -CHECK | ++outline the command line parameters | ++
| -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 | ++
***General settings:**** ++
| Parameter | ++Description | ++
| -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, PIR or NEXUS | ++
| -OUTORDER= | ++INPUT or ALIGNED | ++
| -CASE= | ++LOWER or UPPER (for GDE output only) | ++
| -SEQNOS= | ++OFF or ON (for Clustal output only) | ++
| Parameter | ++Description | ++
| -TOPDIAGS=n | ++number of best diags. | ++
| -WINDOW=n | ++window around best diags. | ++
| -PAIRGAP=n | ++gap penalty | ++
| -SCORE= | ++PERCENT or ABSOLUTE | ++
| Parameter | ++Description | ++
| -PWDNAMATRIX= | ++DNA weight matrix=IUB, CLUSTALW or filename | ++
| -PWGAPOPEN=f | ++gap opening penalty | ++
| -PWGAPEXT=f | ++gap opening penalty | ++
| Parameter | ++Description | ++
| -USETREE= | ++file for old guide tree | ++
| -MATRIX= | ++Protein weight matrix=BLOSUM, PAM, GONNET, ID or filename | ++
| -DNAMATRIX= | ++DNA weight matrix=IUB, CLUSTALW or filename | ++
| -GAPOPEN=f | ++gap opening penalty | ++
| -GAPEXT=f | ++gap extension penalty | ++
| -ENDGAPS | ++no end gap separation pen. | ++
| -GAPDIST=n | ++gap separation pen. range | ++
| -NOPGAP | ++residue-specific gaps off | ++
| -NOHGAP | ++hydrophilic gaps off | ++
| -HGAPRESIDUES= | ++list hydrophilic res. | ++
| -MAXDIV=n | ++% ident. for delay | ++
| -TYPE= | ++PROTEIN or DNA | ++
| -TRANSWEIGHT=f | ++transitions weighting | ++
| Parameter | ++Description | ++
| -NEWTREE1= | ++file for new guide tree for profile1 | ++
| -NEWTREE2= | ++file for new guide tree for profile2 | ++
| -USETREE1= | ++file for old guide tree for profile1 | ++
| -USETREE2= | ++file for old guide tree for profile2 | ++
| Parameter | ++Description | ++
| -NEWTREE= | ++file for new guide tree | ++
| -USETREE= | ++file for old guide tree | ++
| Parameter | ++Description | ++
| -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 | ++
| Parameter | ++Description | ++
| -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 | ++
++
++++ ++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, 25:4876-4882. +
+--- clustalx-1.83.orig/debian/patches/trees.c.patch +++ clustalx-1.83/debian/patches/trees.c.patch @@ -0,0 +1,13 @@ +Index: clustalw-1.83/trees.c +=================================================================== +--- clustalw-1.83.orig/trees.c ++++ clustalw-1.83/trees.c +@@ -1497,7 +1497,7 @@ + fprintf(stdout,"\n or 3) use the PHYLIP package."); + fprintf(stdout,"\n\n"); + if (usemenu) +- getstr("Press [RETURN] to continue",dummy); ++ getstr("Press [RETURN] to continue",10,dummy); + } + + --- clustalx-1.83.orig/debian/patches/series +++ clustalx-1.83/debian/patches/series @@ -0,0 +1,11 @@ +amenu.c.patch +clustal-help.patch +clustalw.h.patch +clustalx.html.patch +interface.c.patch +sequence.c.patch +trees.c.patch +util.c.patch +makefile.patch +clustalx_help.patch +xmenu.c.patch --- clustalx-1.83.orig/debian/patches/clustalx_help.patch +++ clustalx-1.83/debian/patches/clustalx_help.patch @@ -0,0 +1,1529 @@ +Index: clustalw-1.83/clustalx_help +=================================================================== +--- clustalw-1.83.orig/clustalx_help ++++ clustalw-1.83/clustalx_help +@@ -1,4 +1,1524 @@ + ++This is the on-line help file for Clustal X (version 1.81), using the NCBI ++Vibrant Toolkit. ++ ++It should be named or defined as: clustalx_help ++except with MSDOS in which case it should be named ClustalX.HLP ++ ++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. ++ ++ ++ ++ ++>>HELP G << ++ General help for CLUSTAL X (1.8) ++ ++Clustal X is a windows interface for the ClustalW multiple sequence alignment ++program. It provides an integrated environment for performing multiple sequence ++and profile alignments and analysing the results. The sequence alignment is ++displayed in a window on the screen. A versatile coloring scheme has been ++incorporated allowing you to highlight conserved features in the alignment. ++The pull-down menus at the top of the window allow you to select all the ++options required for traditional multiple sequence and profile alignment. ++ ++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.) ++ ++ ++
++ 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 ++++ ++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. ++ ++ ++>>HELP F << ++ 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. 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: ++
++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 T << ++ 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 large number of bootstrap replicates (1000 is ++recommended, even if it means running the program for an hour on a slow ++computer). You can also supply a seed number for the random number generator ++here. Different runs with the same seed will give the same answer. See the ++documentation for more details. ++ ++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). ++ ++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 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. ++ ++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. ++ ++
++ 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 BACKGROUND COLORING option shows the sequence residues using a ++black character on a colored background. It can be switched off to show ++residues as a colored character on a white background. ++ ++Either BLACK AND WHITE or DEFAULT COLOR options can be selected. The Color ++option looks first for the color parameter file (as described above) and, if no ++file is found, uses the default residue-specific colors. ++ ++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. ++ ++
++ -------------------------------------------------------------------- ++@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 ++ ++@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. ++ 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 #. ++ ++ ++>>HELP Q << ++ Alignment Quality Analysis ++ ++
++ 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. ++ ++
++ 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. ++ ++ ++>>HELP 9 << ++ Command Line Parameters ++ ++ DATA (sequences) ++ ++-INFILE=file.ext :input sequences ++-PROFILE1=file.ext and -PROFILE2=file.ext :profiles (aligned sequences) ++ ++ ++ VERBS (do things) ++ ++-OPTIONS :list the command line parameters ++-HELP or -CHECK :outline the command line parameters ++-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 ++ ++ ++ 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, PIR or NEXUS ++-OUTORDER= :INPUT or ALIGNED ++-CASE= :LOWER or UPPER (for GDE output only) ++-SEQNOS= :OFF or ON (for Clustal output only) ++ ++ ++***Fast Pairwise Alignments:*** ++-KTUPLE=n :word size ++-TOPDIAGS=n :number of best diags. ++-WINDOW=n :window around best diags. ++-PAIRGAP=n :gap penalty ++-SCORE= :PERCENT or ABSOLUTE ++ ++ ++***Slow Pairwise Alignments:*** ++-PWMATRIX= :Protein weight matrix=BLOSUM, PAM, GONNET, ID or filename ++-PWDNAMATRIX= :DNA weight matrix=IUB, CLUSTALW or filename ++-PWGAPOPEN=f :gap opening penalty ++-PWGAPEXT=f :gap opening penalty ++ ++ ++***Multiple Alignments:*** ++-NEWTREE= :file for new guide tree ++-USETREE= :file for old guide tree ++-MATRIX= :Protein weight matrix=BLOSUM, PAM, GONNET, ID or filename ++-DNAMATRIX= :DNA weight matrix=IUB, CLUSTALW or filename ++-GAPOPEN=f :gap opening penalty ++-GAPEXT=f :gap extension penalty ++-ENDGAPS :no end gap separation pen. ++-GAPDIST=n :gap separation pen. range ++-NOPGAP :residue-specific gaps off ++-NOHGAP :hydrophilic gaps off ++-HGAPRESIDUES= :list hydrophilic res. ++-MAXDIV=n :% ident. for delay ++-TYPE= :PROTEIN or DNA ++-TRANSWEIGHT=f :transitions weighting ++ ++ ++***Profile Alignments:*** ++-PROFILE :Merge two alignments by profile alignment ++-NEWTREE1= :file for new guide tree for profile1 ++-NEWTREE2= :file for new guide tree for profile2 ++-USETREE1= :file for old guide tree for profile1 ++-USETREE2= :file for old guide tree for profile2 ++ ++ ++***Sequence to Profile Alignments:*** ++-SEQUENCES :Sequentially add profile2 sequences to profile1 alignment ++-NEWTREE= :file for new guide tree ++-USETREE= :file for old guide tree ++ ++ ++***Structure Alignments:*** ++-NOSECSTR1 :do not use secondary structure/gap penalty mask for profile 1 ++-NOSECSTR2 :do not use secondary structure/gap penalty mask for profile 2 ++-SECSTROUT=STRUCTURE or MASK or BOTH or NONE :output in alignment file ++-HELIXGAP=n :gap penalty for helix core residues ++-STRANDGAP=n :gap penalty for strand core residues ++-LOOPGAP=n :gap penalty for loop regions ++-TERMINALGAP=n :gap penalty for structure termini ++-HELIXENDIN=n :number of residues inside helix to be treated as terminal ++-HELIXENDOUT=n :number of residues outside helix to be treated as terminal ++-STRANDENDIN=n :number of residues inside strand to be treated as terminal ++-STRANDENDOUT=n:number of residues outside strand to be treated as terminal ++ ++ ++***Trees:*** ++-OUTPUTTREE=nj OR phylip OR dist OR nexus ++-SEED=n :seed number for bootstraps ++-KIMURA :use Kimura's correction ++-TOSSGAPS :ignore positions with gaps ++-BOOTLABELS=node OR branch :position of bootstrap values in tree display ++ ++ ++>>HELP R << ++ 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 X: ++ ++ ++Jeanmougin,F., Thompson,J.D., Gouy,M., Higgins,D.G. and Gibson,T.J. (1998) ++Multiple sequence alignment with Clustal X. Trends Biochem Sci, 23, 403-5. ++ ++ ++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: ++ ++ ++http://www-igbmc.u-strasbg.fr/BioInfo/ ++ ++ + This is the on-line help file for Clustal X (version 1.83), using the NCBI + Vibrant Toolkit. + --- clustalx-1.83.orig/debian/patches/xmenu.c.patch +++ clustalx-1.83/debian/patches/xmenu.c.patch @@ -0,0 +1,13 @@ +Index: xmenu.c +=================================================================== +--- ./xmenu.c (révision 173) ++++ ./xmenu.c (révision 174) +@@ -4411,7 +4411,7 @@ + while(TRUE) { + if(fgets(temp,MAXLINE+1,fd) == NULL) { + if(!found_help) +- error("No help found in help file"); ++ error("No help found in help file [%s]",help_file); + fclose(fd); + return; + } --- clustalx-1.83.orig/debian/patches/amenu.c.patch +++ clustalx-1.83/debian/patches/amenu.c.patch @@ -0,0 +1,130 @@ +Index: clustalw-1.83/amenu.c +=================================================================== +--- clustalw-1.83.orig/amenu.c ++++ clustalw-1.83/amenu.c +@@ -184,7 +184,7 @@ + fprintf(stdout," H. HELP\n"); + fprintf(stdout," X. EXIT (leave program)\n\n\n"); + +- getstr("Your choice",lin1); ++ getstr("Your choice",MAXLINE+1,lin1); + + switch(toupper(*lin1)) { + case '1': seq_input(FALSE); +@@ -268,7 +268,7 @@ + fprintf(stdout," H. HELP\n"); + fprintf(stdout," or press [RETURN] to go back to main menu\n\n\n"); + +- getstr("Your choice",lin1); ++ getstr("Your choice",MAXLINE+1,lin1); + if(*lin1 == EOS) return; + + switch(toupper(*lin1)) +@@ -361,7 +361,7 @@ + fprintf(stdout," H. HELP\n"); + fprintf(stdout," or press [RETURN] to go back to main menu\n\n\n"); + +- getstr("Your choice",lin1); ++ getstr("Your choice",MAXLINE+1,lin1); + if(*lin1 == EOS) return; + + switch(toupper(*lin1)) +@@ -457,7 +457,7 @@ + fprintf(stdout,"\n\n"); + fprintf(stdout," H. HELP\n\n\n"); + +- getstr("Enter number (or [RETURN] to exit)",lin2); ++ getstr("Enter number (or [RETURN] to exit)",MAXLINE+1,lin2); + if( *lin2 == EOS) { + return; + } +@@ -533,7 +533,7 @@ + fprintf(stdout,"--\n"); + + +- getstr("\n\nEnter number (or [RETURN] to exit)",lin2); ++ getstr("\n\nEnter number (or [RETURN] to exit)",MAXLINE+1,lin2); + if(*lin2 == EOS) return(output_struct_penalties); + + switch(toupper(*lin2)) +@@ -602,7 +602,7 @@ + fprintf(stdout," H. HELP\n"); + fprintf(stdout," or press [RETURN] to go back to main menu\n\n\n"); + +- getstr("Your choice",lin1); ++ getstr("Your choice",MAXLINE+1,lin1); + if(*lin1 == EOS) return; + + switch(toupper(*lin1)) +@@ -677,7 +677,7 @@ + fprintf(stdout,"\n"); + fprintf(stdout," H. HELP\n\n\n"); + +- getstr("Enter number (or [RETURN] to exit)",lin2); ++ getstr("Enter number (or [RETURN] to exit)",MAXLINE+1,lin2); + if(*lin2 == EOS) return; + + switch(toupper(*lin2)) { +@@ -766,7 +766,7 @@ + fprintf(stdout,"\n"); + fprintf(stdout," H. HELP\n\n\n"); + +- getstr("Enter number (or [RETURN] to exit)",lin2); ++ getstr("Enter number (or [RETURN] to exit)",MAXLINE+1,lin2); + if(*lin2 == EOS) return; + + switch(toupper(*lin2)) { +@@ -907,7 +907,7 @@ + + fprintf(stdout," H. HELP\n\n\n"); + +- getstr("Enter number (or [RETURN] to exit)",lin2); ++ getstr("Enter number (or [RETURN] to exit)",MAXLINE+1,lin2); + if( *lin2 == EOS) { + if(dnaflag) { + dna_pw_go_penalty = pw_go_penalty; +@@ -1029,7 +1029,7 @@ + fprintf(stdout," 8. Protein Gap Parameters\n\n"); + fprintf(stdout," H. HELP\n\n\n"); + +- getstr("Enter number (or [RETURN] to exit)",lin2); ++ getstr("Enter number (or [RETURN] to exit)",MAXLINE+1,lin2); + + if(*lin2 == EOS) { + if(dnaflag) { +@@ -1122,7 +1122,7 @@ + fprintf(stdout," 5. Toggle End Gap Separation :%s\n\n",(!use_endgaps) ? "OFF" : "ON"); + fprintf(stdout," H. HELP\n\n\n"); + +- getstr("Enter number (or [RETURN] to exit)",lin2); ++ getstr("Enter number (or [RETURN] to exit)",MAXLINE+1,lin2); + + if(*lin2 == EOS) return; + +@@ -1136,7 +1136,7 @@ + case '3': + fprintf(stdout,"Hydrophilic Residues Currently: %s\n",hyd_residues); + +- getstr("Enter residues (or [RETURN] to quit)",lin1); ++ getstr("Enter residues (or [RETURN] to quit)",MAXLINE+1,lin1); + if (*lin1 != EOS) { + for (i=0;i