CodonLogo 1.0: User's Manual

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Contents

Sequence logo example.

Introduction

CodonLogo is a web based application designed to make the generation of codon sequence logos as easy and painless as possible. It is almost entirely based on the application WebLogo.

Sequence logos are a graphical representation of an amino acid or nucleic acid multiple sequence alignment. Each logo consists of stacks of symbols, one stack for each position in the sequence. The overall height of the stack indicates the sequence conservation at that position, while the height of symbols within the stack indicates the relative frequency of each amino or nucleic acid at that position. The width of the stack is proportional to the fraction of valid symbols in that position. (Positions with many gasp have thin stacks.) In general, a sequence logo provides a richer and more precise description of, for example, a binding site, than would a consensus sequence.

References

Crooks GE, Hon G, Chandonia JM, Brenner SE WebLogo: A sequence logo generator, Genome Research, 14:1188-1190, (2004) [Full Text ]

Schneider TD, Stephens RM. 1990. Sequence Logos: A New Way to Display Consensus Sequences. Nucleic Acids Res. 18:6097-6100

Creating Sequences Logos using the Web interface

Sequence Data

Enter your multiple sequence alignment here, or select a file to upload. Supported file formats include CLUSTALW, FASTA, plain flatfile, MSF, NBRF, PIR, NEXUS and PHYLIP. All sequences must be the same length, else CodonLogo will return an error and report the first sequence that differed in length from previous sequences.

Output format

  • PNG : (600 DPI) Print resolution bitmap
  • PNG : (low res, 96 DPI) Screen resolution bitmap
  • JPEG :Screen resolution bitmap
  • EPS : Encapsulated postscript
  • PDF : Portable Document Format
Generally speaking, vector formats (EPS and PDF) are better for printing, while bitmaps (JPEG and PNG) are more suitable for displaying on the screen or embedding into a web page.

Logo size

The physical dimensions of the generated logo. Specifically, controls the size of individual symbols stacks.
  • small : 5.4 points wide (Same as 9pt Courier), aspect ratio 5:1
  • medium : Double the width and height of small.
  • large : Triple the width and height of small.
The choices have been limited to promote inter-logo consistency. Small logos can fit 80 stacks across a printed page, or 40 across a half page column. The command line interface provides greater control, if so desired.

Stacks per line

If the length of the sequences is greater than this maximum number of stacks per line, then the logo will be split across multiple lines.

Sequence type

The type of biological molecule.
  • auto: Automatically guess sequence type from the data
  • protein
  • dna
  • rna

Ignore lower case

Disregard lower case letters in the and only count upper case letters in sequences?

Units

The units used for the y-axis.
  • probability: Show residue probabilities, rather than information content. If compositional adjustment is disabled, then these are the raw residue frequencies.
  • bits: Information content in bits
  • nats: Natural units, 1 bit = ln 2 (0.69) nats
  • kT : Thermal energy units in natural units (Numerically the same as nats)
  • kJ/mol : Thermal energy (Assuming T = 300 K)
  • kcal/mol : Thermal energy (Assuming T = 300 K)

First position number

The numerical label of the first residue in the multiple sequence alignment. The label must be an integer. Residue labels for the logo will be relative to this number. (See also: Logo Range)

Logo range

By default, all sequence data is displayed in the Sequence Logo. With this option, you can instead show a subrange of the entire sequence. Start and end positions are included, and the numbering of positions is relative to the sequence number of the first position. (See also: First Position Number ) Thus, if the first position number is "2", start is "5" and end is "10", then the 4th through 9th (inclusive) sequence positions will be displayed, and they will be numbered "5", "6", "7", "8", "9" and "10".

Composition

The background composition of the genome or proteome from which the sequences have been drawn. The default, automatic option is to use equiprobable background for nucleic acids and a typical amino acid usage pattern for proteins. However, you may also explicitly set the expected CG content for nucleic acid sequences, insists on equiprobable background distributions, or turn off composition adjustment altogether.

Compositional adjustment has two effects. First, the information content of a site is defined as the relative entropy of the monomers at that site to the background distribution. Consequentially, rare monomers have higher information content (when they occur) than relatively common monomers.

Secondly, the background composition is used in the small sample correction of information content. Briefly, if only a few sequences are available in the multiple sequence alignment, then sites typically appear more conserved than they really are. Small samples bias the relative entropy upwards. To compensate, we add pseudocounts to the actual counts, proportional to the expected background composition. These pseudocounts smooth the data for small samples, but become irrelevant for large samples. The proportionality constant is set to 4 for nucleic acid sequences, and 20 for proteins (These numbers have been found to give reasonable results in practice).

Behind the scenes, things are more complex. We do a full Bayesian calculation, starting with explicit Dirichlet priors based on the background composition, to which we add the data and then calculate both the posterior mean relative entropy (the stack height) and Bayesian 95% confidence intervals for error bars. These interesting details will be explained elsewhere.

Scale stack width

Scale the visible stack width by the fraction of symbols in the column? (i.e. columns with many gaps or unknown residues are narrow.)

Error bars

Display error bars. These indicate an approximate, Bayesian 95% confidence interval.

Title

Give your logo a title.

Figure label

An optional figure label, added to the top left (e.g. '(a)')

X-axis

Add a label to the x-axis, or hide axis altogether.

Y-axis

The vertical axis indicates the information content of a sequence position. Use this option to toggle the y-axis and override the default axis label.

Y-axis scale

The height of the y-axis in designated units. The automatic option will pick reasonable defaults based on the sequence type and axis unit.

Y-axis tic spacing

The distance between major tic marks on the Y-axis.

Sequence end labels

Choose this option to label the 5' & 3' ends of nucleic acid or the N & C termini of amino acid sequences.

Version fineprint

Toggle display of the CodonLogo version information in the lower right corner.

Color Scheme

  • auto : use Base Pairing for nucleic acids (NA), Hydrophobicity for amino acids (AA).
  • monochrome: All symbols black
  • Base Pairing (NA default) :
    2 Watson-Crick hydrogen bondsTAUdark orange
    3 Watson-Crick hydrogen bondsGC blue
  • Classic (NA) : WebLogo (version 1 and 2) and makelogo default color scheme for nucleic acids: G, orange; T & U, red; C, blue; and A, green.
    G orange
    TU red
    Cblue
    Agreen
  • Hydrophobicity (AA default) :
    HydrophobicRKDENQ black
    NeutralSGHTAP green
    HydrophilicYVMCLFIWblue
  • Chemistry (AA): Color amino acids according to chemical properties. WebLogo (version 1 and 2) and makelogo default color.
    PolarG,S,T,Y,C,Q,N green
    BasicK,R,H blue
    AcidicD,Ered
    HydrophobicA,V,L,I,P,W,F,Mblack
  • Charge (AA) :
    PositiveKRH blue
    NegativeDE red
  • Custom : A custom color scheme can be specified in the input field below. Specify colors on the left and associated symbols on the right. Colors are entered using CSS2 (Cascading Style Sheet) syntax. (e.g. 'red', '#F00', '#FF0000', 'rgb(255, 0, 0)', 'rgb(100%, 0%, 0%)' or 'hsl(0, 100%, 50%)' for the color red.)

More Options

The CodonLogo command line client, codonlogo, provides many more options and greater control over the final logo appearance.

Installing CodonLogo

Dependencies

CodonLogo is written in python. It is necessary to have python 2.3, 2.4 or 2.5 and the extension packages numpy and corebio installed before WebLogo will run. WebLogo also requires a recent version of ghostscript to create PNG and PDF output.

After unpacking the CodonLogo tarfile, it should be possible to immediately create logos using the command line client (Provided that python, numpy, corebio and ghostscript have already been installed).

 
./codonlogo --format PNG < htdocs/examples/cap_hth.fa > cap_hth.png   

Please consult the file build_examples.sh for more examples.

To run CodonLogo as a stand alone web service, run the logo server command :

 
./codonlogo --serve 

It should now be possible to access CodonLogo at http://localhost:8080/.

The command line client and WebLogo libraries can be permanently installed using the supplied setup.py script.

 
sudo python setup.py install
Run python setup.py help for more installation options. For example, to specifically install the CodonLogo script to /usr/local/bin
sudo python setup.py install_scripts --install-dir /usr/local/bin

Web App

To use CodonLogo as a web application, first install the weblogo dependancies and libraries as above, then place (or link) the weblogolib/weblogo_htdocs directory somewhere within the document root of your webserver. The webserver must be able to execute the CGI script create.cgi. For Apache, you may have to add an ExecCGI option and add a cgi handler in the httpd.conf configuration file. Something like this:

<Directory "/home/httpd/htdocs/weblogo/">
    Options FollowSymLinks MultiViews ExecCGI
    AllowOverride None
    Order allow,deny
    Allow from all
</Directory>
...
# To use CGI scripts outside of ScriptAliased directories:
# (You will also need to add "ExecCGI" to the "Options" directive.)
#
AddHandler cgi-script .cgi
It may also be necessary to set the PATH and PYTHONPATH environment variables.
SetEnv PYTHONPATH /path/to/weblogo/libraries
The cgi script also has to be able to find the 'gs' ghostscript executable. If ghostscipt is installed in a non-standard location add the following environment variable.
SetEnv COREBIOPATH /path/to/gs
The maxium bytes of uploaded sequecne data can be controlled with the WEBLOGO_MAX_FILE_SIZE environment variable.
SetEnv WEBLOGO_MAX_FILE_SIZE 1000000

codonlogo, The CodonLogo Command Line Interface (CLI)

The command line client has many options not available through the web interface. Please consult the bundled build_examples.sh script for inspiration.
	Usage: codonlogo [options]  < sequence_data.fa > sequence_logo.eps

	Create sequence logos from biological sequence alignments.

	Options:
	     --version                  show program's version number and exit
	  -h --help                     show this help message and exit

	  Input/Output Options:
	    -f --fin FILENAME           Sequence input file (default: stdin)
	       --fin-format FORMAT      Multiple sequence alignment format: (clustal,
	                                fasta, plain, msf, genbank, nbrf, nexus,
	                                phylip, stockholm, intelligenetics, table,
	                                array)
	    -o --fout FILENAME          Output file (default: stdout)
	    -F --format FORMAT          Format of output: eps (default), png,
	                                png_print, pdf, jpeg, txt

	  Logo Data Options:
	    -A --sequence-type TYPE     The type of sequence data: 'protein', 'rna' or
	                                'dna'.
	    -a --alphabet ALPHABET      The set of symbols to count, e.g. 'AGTC'. All
	                                characters not in the alphabet are ignored. If
	                                neither the alphabet nor sequence-type are
	                                specified then weblogo will examine the input
	                                data and make an educated guess. See also
	                                --sequence-type, --ignore-lower-case
	       --ignore-lower-case YES/NO
	                                Disregard lower case letters and only count
	                                upper case letters in sequences? (Default: No)
	    -U --units NUMBER           A unit of entropy ('bits' (default), 'nats',
	                                'digits'), or a unit of free energy ('kT',
	                                'kJ/mol', 'kcal/mol'), or 'probability' for
	                                probabilities
	       --composition COMP.      The expected composition of the sequences:
	                                'auto' (default), 'equiprobable', 'none' (Do
	                                not perform any compositional adjustment), a
	                                CG percentage, a species name (e.g. 'E. coli',
	                                'H. sapiens'), or an explicit distribution
	                                (e.g. {'A':10, 'C':40, 'G':40, 'T':10}). The
	                                automatic option uses a typical distribution
	                                for proteins and equiprobable distribution for
	                                everything else.
	       --weight NUMBER          The weight of prior data.  Default: total
	                                pseudocounts equal to the number of monomer
	                                types.
	    -i --first-index INDEX      Index of first position in sequence data
	                                (default: 1)
	    -l --lower INDEX            Lower bound of sequence to display
	    -u --upper INDEX            Upper bound of sequence to display

	  Logo Format Options:
	    These options control the format and display of the logo.

	    -s --size LOGOSIZE          Specify a standard logo size (small, medium
	                                (default), large)
	    -n --stacks-per-line COUNT  Maximum number of logo stacks per logo line.
	                                (default: 40)
	    -t --title TEXT             Logo title text.
	       --label TEXT             A figure label, e.g. '2a'
	    -X --show-xaxis YES/NO      Display sequence numbers along x-axis?
	                                (default: True)
	    -x --xlabel TEXT            X-axis label
	    -S --yaxis UNIT             Height of yaxis in units. (Default: Maximum
	                                value with uninformative prior.)
	    -Y --show-yaxis YES/NO      Display entropy scale along y-axis? (default:
	                                True)
	    -y --ylabel TEXT            Y-axis label  (default depends on plot type
	                                and units)
	    -E --show-ends YES/NO       Label the ends of the sequence? (default:
	                                False)
	    -P --fineprint TEXT         The fine print (default: weblogo version)
	       --ticmarks NUMBER        Distance between ticmarks (default: 1.0)
	       --errorbars YES/NO       Display error bars? (default: True)

	  Color Options:
	    Colors can be specified using CSS2 syntax. e.g. 'red', '#FF0000', etc.

	    -c --color-scheme SCHEME    Specify a standard color scheme (auto, base
	                                pairing, charge, chemistry, classic,
	                                hydrophobicity, monochrome)
	    -C --color COLOR SYMBOLS DESCRIPTION 
	                                Specify symbol colors, e.g. --color black AG
	                                'Purine' --color red TC 'Pyrimidine'
	       --default-color COLOR    Symbol color if not otherwise specified.

	  Advanced Format Options:
	    These options provide fine control over the display of the logo.

	    -W --stack-width POINTS     Width of a logo stack (default: 10.8)
	    -H --stack-height POINTS    Height of a logo stack (default: 54.0)
	       --box YES/NO             Draw boxes around symbols? (default: no)
	       --resolution DPI         Bitmap resolution in dots per inch (DPI).
	                                (default: 96 DPI, except png_print, 600 DPI)
	                                Low resolution bitmaps (DPI<300) are
	                                antialiased.
	       --scale-width YES/NO     Scale the visible stack width by the fraction
	                                of symbols in the column?  (i.e. columns with
	                                many gaps of unknowns are narrow.)  (default:
	                                yes)
	       --debug YES/NO           Output additional diagnostic information.
	                                (default: False)

	  CodonLogo Server:
	    Run a standalone webserver on a local port.

	       --serve                  Start a standalone CodonLogo server for creating
	                                sequence logos.
	       --port PORT              Listen to this local port. (Default: 8080)

WebLogo Application Programmer Interface (API)

The WebLogo python libraries provide even greater flexibility than the command line client. The code is split between two principle packages, weblogo itself, which contains specialized sequence logo generation code, and corebio, a package that contains code of more general utility. Please consult the WebLogo and CoreBio API documentation.

WebLogo Development and Future Features

The development project is hosted at http://code.google.com/p/weblogo. If you wish to extend WebLogo or to contribute code, then you should download the full source code development package directly from the subversion repository.

> svn checkout http://weblogo.googlecode.com/svn/trunk/ weblogo
>  cd weblogo
> ./weblogo < cap.fa > cap.eps

Please consult the developer notes, DEVELOPERS.txt and software license LICENSE.txt

Outstanding bugs and feature requests are listed on the WebLogo issue tracker.

Miscellanea

Release Notes and Known Bugs

The WebLogo release notes detail changes to WebLogo and known issues with particular versions.

WebLogo 2

The legacy WebLogo 2 sever can be found here.

Acknowledgments

WebLogo was created by Gavin E. Crooks, Liana Lareau, Gary Hon, John-Marc Chandonia and Steven E. Brenner. Many others have provided suggestions, bug fixes and moral support.

WebLogo was originally based upon the programs alpro and makelogo, both of which are part of Tom Schneider's delila package. Many thanks are due to him for making this software freely available and for encouraging its use.