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BOXSHADE(1) Boxshade Manual BOXSHADE(1)


boxshade - Pretty-printing of multiple sequence alignments




BOXSHADE is a program for pretty-printing multiple alignment output. The program itself doesn't do any alignment, you have to use a multiple alignment program like ClustalW or Pileup and use the output of these programs as input for BOXSHADE.


Show the help.


Show the help and extend command line.


Use defaults, no unnecessary questions.


Use default numbering.


Assume DNA sequences, use box_dna.par.


Create separate files for multiple pages.


Shading according to sequence No. xxx.


xxxxx is input file name.


xxxxx is output file name.


xxxxx is parameter file name.


xxxxx is file name for similar residues def.


xxxxx is file name for grouping residues def.


x is the fraction of sequences that must agree for a consensus.


x is output device class (see below).


x is input file format (see below).


Print ruler line.


Create consensus line.


xyz are consensus symbols.


If the one above does not work, try this one.


Output files lines are terminated with LF only.


Output files lines are terminated with CR only.


Output files lines are terminated with CRLF.

This manual page was written for the Debian(TM) distribution because the original program does not have a manual page. The presented information comes from the documentation of the Web Service of the 3.21 version that is not available as a Debian package.

BOXSHADE is a program for creating good looking printouts from multiple-aligned protein or DNA sequences. The program does no alignment by itself, it has to take as input a file preprocessed by a multiple alignment program or a multiple file editor. See below for a list of supported input formats and output devices. In the standard BOXSHADE output, identical and similar residues in the multiple-alignment chart are represented by different colors or shadings. There are some more options concerning the kind of shading to be applied, sequence numbering, consensus output and so on. The user interface is a bit clumsy at the moment, one has to answer a lot of questions in order to get the desired output. There is, however, the possibility to use default parameters from a standard parameter file or to supply the program with parameters from the command line. At the moment, the VMS and DOS versions of BOXSHADE have identical user interfaces.

Input formats

BOXSHADE 3.2 knows about the following input file formats: (some of the are generally used only for MSDOS or VMS systems) + CLUSTAL and CLUSTALV, multiple alignment program, DOS/VMS/MAC default extension .ALN + ESEE, multiple sequence editor, DOS default extension .ESE + PHYLIP, phylogenetic analysis package, DOS, VMS, UNIX default extension .PHY + PILEUP and PRETTY of the GCG sequence analysis package VMS/UNIX default extensions .MSF and .PRE NB!! you are strongly encouraged NOT to use the PRETTY format as input, it may be incompatible with the revised version of .MSF input. We can't actually think why anyone would use this format now, .MSF files are more useful generally. + MALIGNED, multiple sequence editor, VMS only default extension .MAL BOXSHADE tries to determine the file type from the extension but will work also if different extensions are used.

Output devices

POSTSCRIPT/EPS creates POSTSCRIPT(TM) files for printing on a Laserprinter or for further conversion with a POSTSCRIPT interpreter (like GHOSTSCRIPT) + HPGL for export to various graphics programs or for conversion/printing with the shareware program PRINTGL. Plotting BOXSHADE output on a plotter is generally not recommended + RTF for export to various word-processing and graphics programs + CRT, uses direct screen writes to the PC-monitor. Possible options depend on the graphics adapter used. This output device is supported only in the MSDOS version. + ANSI. On a PC, this option uses an ANSI device driver (ANSI.SYS) that has to be loaded in CONFIG.SYS previously. Possible character renditions are reverse, bold,underlined, blinking etc. On non-DOS systems, this option behaves more or less like the VT100 output mode. + VT100 for display on a VT100 compatible terminal or emulator. + ReGISterm for display on a ReGIS compatible graphics terminal or emulator. + ReGISfile for later conversion by the program RETOS (copyright DEC) in order to print on DIGITALs printer series. + LJ250 for printing on DIGITALS LJ250 color printer. + ASCII output showing either the conserved residues or the varying ones (others as '-'). + FIG file for xfig 2.1. + PICT files for import to Mac and PC graphics progs. Some of the formats above offer the possibility of scaling the characters and of rotating the plot. Character size has to be entered in 'point' units. Normal output orientation is in portrait mode (PS/EPS/HPGL/PICT only), to obtain output in landscape orientation, 'rotate plot = y' has to be chosen. When creating multi-page output, all pages are contained in a single output file. If one page per file is desired, one has to use the command line parameter /SPLIT. This is enforced when requesting EPSF or PICT file output, as multi-page EPSFs are a contradiction of the purpose of an EPSF and large PICT files would probably be too big for most personal computers. While using the terminal as output device, the 'RETURN' key has to be pressed to obtain the next page of output.

Sequence numbering

Starting with version 2.2 there is the possibility to add numbering to the output files. The numbers are printed between the sequence names and the sequence itself. Since most of the input-files either use no numbering or number the first position in the alignment always with a "1" (and that does not necessarily reflect the numbers within the original sequence), the user is asked to enter the starting position for each sequence. The command line flag /DEFNUM suppressed that question, a starting position of 1 is assumed for all sequences. Boxshade starts with the value entered for the leftmost position and continues numbering every valid symbol, skipping blanks, '-','.' and stuff like that.

Default parameters

Several people using previous releases of BOXSHADE pointed me to the need of having default parameters for the various questions asked by the program. They argued that most sites only use one type of input files, one output device and one choice of colors for the output. I therefore added a management of default parameters allowing two levels of assistance to the user. 1) all default parameters are contained in an ASCII file that can be modified easily to accommodate the users taste. The format is roughly documented within the file-header, it resembles the keyboard input one has to make if using the program interactively. There are two such files supplied with this release of BOXSHADE, BOX_DNA.PAR and BOX_PEP.PAR , holding some example parameters for peptide and dna-comparisons. there are no big differences between these two, the major one is that when shading DNA-comparisons one doesn't care of "similar" residues. 2) to run the program with minimal user interaction, I have added the possibility to use command line parameters. At the moment, you can use: /check : list all allowed command line parameters (this list) and allows parameters to be added. /def : program runs without questions, BOX_PEP.PAR is used as default /dna : makes the program use BOX_DNA.PAR as parameter file /pep : makes the program use BOX_PEP.PAR as parameter file /in=xxx : makes the program take xxx as input file /out=yyy : makes the program take yyy as output file (note1) /par=zzz : makes the program use zzz as a default parameter file /type=1 : makes the program assume an input file of type 1 (PRETTY/MSF) /dev=1 : makes the program assume and output device of type 1 (CRT) /numdef : use default numbering (all sequences starting with "1") /thr : threshold fraction of residues that must agree for a consensus /split : forces one page per file output, creates multiple output files. /cons : makes the program create an additional consensus line (see below) /symbcons=: influences the way the consensus line is displayed. (see below) /unix : writes output files in unix style (LF only) (note2) /dos : writes output files in DOS style (CR/LF) (note2) note1: on unix machines, use out=OUTPUT for terminal output on DOS machines, use out=con: on VMS machines, use out=tt: note2: if no mode is specified, the native style of the machine is used.


on unix systems, the dash (-) instead of the slash (/) has to be used as separation character for command line parameters. For example, a valid unix command line is: boxshade -def -numdef -cons -symbcons=" .*"

Shading strategies (similarity to consensus or single sequence)

Starting with version 3, BOXSHADE has a new shading system. The first difference is the introduction of a threshold fraction of residues that must agree for there to be a consensus. Previously, the program assumed that SOME residue was always the consensus. If no two residues were the same, the first sequence provided the consensus residue. This threshold fraction can be any number between 0.0 and 1.0. The number of sequences that must agree for there to be a consensus is, as you might expect, this fraction times the total number of sequences in the alignment (fractions of a sequence count as one, e.g. 3.2 becomes 4). The second difference is the idea of 'consensus by similarity'; this tries to take account of the situations where all the sequences may have (for example) R or K at a position, but neither in a majority. It would not be logical to shade one type of residue as 'identical' and the other as 'similar'; the threshold function might also eliminate both as being in too small numbers. Therefore, if there is not a single residue that is conserved (greater than the threshold) at a position, the program looks for a 'group' of amino acids that fulfills the requirements. 'Groups' are defined in the .grp files. Users can tailor these to their personal prejudices. Any amino acid not listed is assumed not to be in a group. All members of a group are considered to be mutually similar, unlike the .sim files, described below. If consensus by similarity is found, all the residues in the consensus are shaded using the 'similar' shading defined by the user. If the user does not select 'shading by similarity', only identity-type consensus is looked at. If an identity-type consensus is found, and similarity shading is in operation, the program looks to see if the remaining residues are similar to the consensus residue. Here the box_xxx.sim files are used. The main difference between relationships in these files and those in the .grp files is that, e.g. in a .grp file the line STA means that all three a.a.s are mutually similar. In a .sim file S TA means that both T and A are considered similar to S, where there is a conserved S residue in more than threshold number of sequences. However, it does NOT mean that T and A are similar to each other. Note that cases where two residues, or groups of residues, fulfill the threshold requirements (as could happen with values of the thr. fraction less than or equal to 0.5) are treated as having no consensus. This describes the main shading model 'shading according to a consensus'. The alternative model is called 'shading according to a master sequence'. In this case the user is prompted for a sequence of the alignment and consecutively that sequence is taken to be the 'consensus'. Only those residues become shaded that are identical or similar to the chosen sequence. Output obtained with this option tends to be less shaded and neglects similarities between the other (non-chosen) sequences. Starting in V2.7, this 'master sequence' can be hidden. Thus, it only influences the shading of the other sequences without being shown itself.

Consensus display

Starting with version 2.5, BOXSHADE offers the possibility to create an additional line holding a consensus symbol. This line can either be obtained by using the command line qualifier /CONS or interactively by answering the question ' create consensus? '. The way this consensus line is displayed can be modified by the command line parameter SYMBCONS=xyz, by editing the respective entry in the .PAR file or interactively. Since the SYMBCONS syntax is not intuitive, here a brief description: The SYMBCONS parameter consist of exactly three symbols: + the first one stands for 'normal' sequence residues that are not involved in any similar/identical relationship. + the second symbol represents positions that are similar in all sequences of the alignment. See the files BOX_PEP.SIM and BOX_DNA.SIM to see what residues are considered similar. + the third symbol represents positions that are identical in all sequences of the alignment. A SYMBCONS parameter string " .*" (blank/point/asterisk) means: label all positions in the alignment with totally identical residues by an asterisk, all positions with all similar residues by a point and do not mark the other positions. The letter 'B' can be used instead of the blank, this is necessary e.g. when using the command line option /SYMBCONS=B.* which gives the same result as the above example. The option /SYMBCONS= .* would result in an unexpected behaviour because MSDOS squeezes blanks out of the command line. Besides points, asterisks and other symbols, there are two special characters when they appear in the SYMBCONS string: 'L' and 'U'. An 'L' means, that a lowercase representation of the most abundant residue at that position is to be used instead of a fixed consensus symbol while an 'U' means an uppercase character representation of that residue. A possible application would be the SYMBCONS string " LU" where similar residues are represented by lowercase characters and identical by uppercase characters.

Shareware/PD programs useful in conjunction with BOXSHADE

multiple alignment files that to be used by BOXSHADE can be created, amongst others, by the following PD/freeware programs: + PHYLIP by Joe Felsenstein, available by ftp from + ESEE by Eric Cabot, available from the same sources as BOXSHADE (see above) + CLUSTAL by Des Higgins, ditto for preview/conversion of POSTSCRIPT files, the program GHOSTSCRIPT from GNU software foundation is highly recommended. It is available from all major MSDOS ftp-sites (e.g. SIMTEL or There is also a version tested for use with boxshade available at although this might be not the most recent release. for Mac users, there is MacGhostscript, also available from the main archives (info-mac, umich and their mirrors). A *very* good tool for putting a preview image into an EPSF file, often a prerequisite for incorporating into a drawing package, is PS2EPS, by Peter Lerup. This can be found on info-mac. for preview/conversion of HPGL files, the shareware program PRINTGL 1.18 by Cary Ravitz is highly recommended. It is available from many MSDOS ftp sites and from - output on dot printers - Since PRINTGL offers a broad choice of printer types and is a nice program, I recommend its use for printing BOXSHADE output on non-POSTSCRIPT printers. Use HPGL output with options 0F1N for normal residues 2F1N for identical residues 3F1N for similar residues 2F4N for conserved residues 8 for character size not rotated (these are the standard parameters in BOX_PEP.PAR) for creating a HPGL files. (lets call it TEST.PLT) Now use PRINTGL either interactively by calling PMI or use a command line like: PRINTGL /Fx/S0340/Waaac/Ptest.plt where test.plt is to be replaced by the filename to convert and the x in the expression /Fx is to be replaced by the letter of the printer you use. (See the PRINTGL documentation for further details)


The RTF output and PHYLIP input implementations are still experimental. Please tell me of your experiences with the program. + the current DOS version supports only 13 sequences with 2000 residues each. This parameters can be easily changed in the source code. If you cannot compile the sources because you are lacking a pascal compiler, contact the author for precompiled versions


There is no publication on BOXSHADE and none is planned. Most people just use it for figures in publications and don't mention anything, this is ok for the authors of BOXSHADE. If you really feel like mentioning BOXSHADE, you could either acknowledge it in the figure legend or in the Mat&Meth part on sequence analysis.



seaview(1) kalign(1)


Kay Hofmann <>
ISREC, Bioinformatics Group,
Epalinges s/Lausanne Switzerland

Wrote Boxshade.

Michael Baron <>
BBSRC Institute for Animal Health,
GU24 0NF

Wrote Boxshade.

Harmut Schirmer <>
Technische Fakultaet,
Kaiserstr. 2

C port of Boxshade. (don't send Kay or Michael any questions concerning the 'C' version of boxshade)

Steffen Möller <>

Wrote the manpage.

Charles Plessy <>

Updated the manpage


Copyright © 1997 Kay Hofmann, Michael Baron and Harmut Schirmer
Copyright © 2003, 2007 Steffen Moeller, Charles Plessy

The above copyright notices refer to the program and its manpage respectively.

BOXSHADE is completely public-domain and may be passed around and modified without any notice to the authors.

This manual page was written for the Debian(TM) system but may be used by others. Permission is granted to copy, distribute and/or modify this document under same terms as boxshade itself.

February 9, 2013 boxshade 3.3.1