WebLogo 3 : User's Manual

Contents

• Introduction
• Creating Sequences Logos using the Web interface
• Downloading and Installing WebLogo
• Command Line Interface (CLI)
• Application Programmer Interface (API)
• Development and Future Features
• Miscellanea

Release Notes and Known Bugs

Introduction

WebLogo is a web based application designed to make the generation of sequence logos as easy and painless as possible.

A sequence logo is 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 gaps 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

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
• SVG : Scalable Vector Graphics

Logo size

• 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.

Stacks per line

Sequence type

• auto: Automatically guess sequence type from the data
• protein
• dna
• rna

Ignore lower case

Units

• 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

Logo range

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, insist 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

Error bars

Title

Figure label

X-axis

Y-axis

Y-axis scale

Y-axis tic spacing

Sequence end labels

Version fineprint

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 bonds	TAU	dark orange
  3 Watson-Crick hydrogen bonds	GC	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
  C	blue
  A	green

• Hydrophobicity (AA default) :

  Hydrophilic	RKDENQ	blue
  Neutral	SGHTAP	green
  Hydrophobic	YVMCLFIW	black

• Chemistry (AA) : Color amino acids according to chemical properties. WebLogo (version 1 and 2) and makelogo default color. (Note that the WebLogo 2 documentation erroneously lists Q and N under green.)

  Polar	G,S,T,Y,C	green
  Neutral	Q,N	purple
  Basic	K,R,H	blue
  Acidic	D,E	red
  Hydrophobic	A,V,L,I,P,W,F,M	black

• Charge (AA) :

  Positive	KRH	blue
  Negative	DE	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

Installing WebLogo

Dependencies

WebLogo version 3 is written in python. It is necessary to have python 2.5, 2.6 or 2.7 and the extension package numpy installed before WebLogo will run. WebLogo also requires a recent version of ghostscript to create PNG and PDF output, and pdf2svg to generate SVG output.

Download and Installation

If the python package setuptools has been installed, then WebLogo and its dependencies can be downloaded and installed with a single command:

sudo easy_install weblogo

Alternatively, weblogo and its dependencies can be installed manually. The WebLogo source code can be downloaded from http://code.google.com/p/weblogo/. This code is distributed under various open source licenses. Please consult the LICENSE.txt file in the source distribution for details.

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

./weblogo --format PNG < cap.fa > cap.png   

Please consult the file build_examples.sh for more examples.

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

./weblogo --serve 

It should now be possible to access WebLogo 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 weblogo script to /usr/local/bin

sudo python setup.py install_scripts --install-dir /usr/local/bin

Web App

To use WebLogo as a web application, first install the weblogo dependencies and libraries as above, then place (or link) the weblogolib/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

SetEnv PYTHONPATH /path/to/weblogo/libraries

SetEnv COREBIOPATH /path/to/gs

SetEnv WEBLOGO_MAX_FILE_SIZE 1000000

weblogo, The WebLogo Command Line Interface (CLI)

Usage: weblogo [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)
    -D --datatype FORMAT        Type of multiple sequence alignment or
                                position weight matrix file: (clustal, fasta,
                                plain, msf, genbank, nbrf, nexus, phylip,
                                stockholm, intelligenetics, table, array,
                                transfac)
    -o --fout FILENAME          Output file (default: stdout)
    -F --format FORMAT          Format of output: eps (default), png,
                                png_print, pdf, jpeg, svg, logodata

  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
    -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 depends on
                                alphabet length
    -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

  Transformations:
    Optional transformations of the sequence data.

       --ignore-lower-case      Disregard lower case letters and only count
                                upper case letters in sequences.
       --reverse                reverse sequences
       --complement             complement nucleic sequences
       --revcomp                reverse complement nucleic sequences

  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
       --annotate TEXT          A comma separated list of custom stack
                                annotations, e.g. '1,3,4,5,6,7'.  Annotation
                                list must be same length as sequences.
    -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)
       --reverse-stacks YES/NO  Draw stacks with largest letters on top?
                                (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)
       --aspect-ratio POINTS    Ratio of stack height to width (default: 5)
       --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)

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

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

WebLogo Application Programmer Interface (API)

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

WebLogo 2

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.

Disclaimer

While no permanent records are kept of submitted sequences, we cannot undertake to guarantee that data sent to WebLogo remains secure. Moreover, no guarantees whatsoever are provided about data generated by WebLogo.

Feedback

Suggestions on how to improve WebLogo are heartily welcomed! Please direct questions to the WebLogo discussion group.