Nucleic Acids Research 2004 32(Web Server Issue):W660-W664; doi:10.1093/nar/gkh410
© 2004, the authors
Nucleic Acids Research, Vol. 32, Web Server issue © Oxford University Press 2004; all rights reserved
PlasMapper: a web server for drawing and auto-annotating plasmid maps
Xiaoli Dong,
Paul Stothard,
Ian J. Forsythe and
David S. Wishart*
Departments of Biological Sciences and Computing Science, University of Alberta, Edmonton, AB, T6G 2E8, Canada
* To whom correspondence should be addressed. Tel: +1 780 492 0383; Fax: +1 780 492 5305; Email: david.wishart{at}ualberta.ca
Received February 15, 2004; Revised and Accepted March 30, 2004
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ABSTRACT
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PlasMapper is a comprehensive web server that automatically
generates and annotates high-quality circular plasmid maps.
Taking only the plasmid/vector DNA sequence as input, PlasMapper
uses sequence pattern matching and BLAST alignment to automatically
identify and label common promoters, terminators, cloning sites,
restriction sites, reporter genes, affinity tags, selectable
marker genes, replication origins and open reading frames. PlasMapper
then presents the identified features in textual form and as
high-resolution, multicolored graphical output. The appearance
and contents of the output can be customized in numerous ways
using several supplied options. Further, PlasMapper images can
be rendered in both rasterized (PNG and JPG) and vector graphics
(SVG) formats to accommodate a variety of user needs or preferences.
The images and textual output are of sufficient quality that
they may be used directly in publications or presentations.
The PlasMapper web server is freely accessible at
http://wishart.biology.ualberta.ca/PlasMapper.
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INTRODUCTION
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Plasmid map generation is one of the oldest and most frequently
performed operations in bioinformatics. Indeed, probably almost
every practicing molecular biologist has worked with or generated
a plasmid map to guide them through the cloning or plasmid manipulation
process. Because of the size and complexity of plasmid molecules,
computer-generated maps are absolutely essential to identify,
locate and analyze key regions in a vector sequence. As early
as the 1980s standalone computer programs were being described
that supported the presentation and manipulation of plasmid
maps on specific platforms and computer operating systems (
1–
7).
Many of these early freeware packages have since been replaced
by more sophisticated and far more user-friendly commercial
packages such as SimVector (Premier BioSoft), GeneTool (BioTools),
VectorNTI (Informax, Invitrogen), MacVector (Accelrys), DNA
Strider and LaserGene (DNAStar). Currently there are remarkably
few freeware plasmid mapping programs still available, although
pDRAW32 (AcaClone) is one example of an installable standalone
package that supports plasmid mapping.
With the growing trend toward using freeware and freely available web tools in bioinformatics, it seems that the continuing dependency on expensive commercial packages to perform just a single operation (plasmid mapping) is somewhat questionable. Furthermore, with the increasing diversity of operating systems seen in many laboratories and the expanding level of inter-laboratory and international collaboration, we believe that a platform-independent solution to plasmid mapping is needed. One obvious solution is a plasmid mapping web server.
Here we describe a web server, called PlasMapper, which is able to accept FASTA-formatted DNA sequences and generate a fully labeled/annotated plasmid map (both graphical and textual output) with essentially no further user input. A central innovation in PlasMapper is its capacity to automatically identify and label the plasmid control sequences found in both eukaryotic and prokaryotic vectors using a large database of common plasmid sequences and common plasmid subsequences (replication origins, promoters, terminators, marker genes, etc.). PlasMapper supports a wide range of textual and visual display options that allow users to easily customize the image or textual output. It is able to generate plasmid maps of sufficient quality and resolution that they may be readily used in publications or presentations. PlasMapper is specifically designed to make plasmid annotation trivially simple and to facilitate the sharing and dissemination of plasmid images and plasmid data across all computer platforms.
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PROGRAM DESCRIPTION
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PlasMapper is composed of three parts: a front-end web interface
(generated using Java), a back-end for rendering and sequence
matching (written in Java and C) and a Feature Site Database
(FSD) consisting of 336 DNA sequence motifs (promoters, terminators,
selectable markers, etc.;
Table 1) and 457 restriction enzymes
from the Restriction Enzyme Database (
8). The FSD was compiled
from an extensive survey of commercially and publicly available
plasmids. PlasMapper accepts FASTA DNA sequences up to 20 000
bases in length as input to its sequence window and performs
checks on both the length and validity of the DNA sequence prior
to conducting any further analysis. To facilitate the generation
of maps for commonly used plasmids, the PlasMapper website also
maintains a growing repository of 288 vector sequences available
from various vendors and suppliers. These sequences may be selected
and automatically uploaded into the sequence window using the
‘Plasmid Library’ button. To facilitate tracking,
editing and ‘virtual cloning’ the sequence text
box has a ‘(Re)Format’ button that allows the raw
FASTA sequence file to be block formatted and numbered. Insertions
(cloned genes), deletions, mutations, edits and corrections
can all be made readily in this specially formatted view. As
soon as any edits are completed, the user can press the ‘(Re)Format’
button to reformat and renumber the modified sequence. After
a sequence has been pasted, edited, selected or reformatted,
the ‘Submit’ button can be used to begin the map
generation process. As seen in
Figure 1, several options are
available for specifying which features are displayed and how
the map image is rendered. More detailed descriptions of how
annotation, labeling and rendering can be controlled are provided
in the on-line Help page.
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Table 1. Features in each category of the Feature Site Database consists of 10 different feature categories and consists of 336 total features
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Figure 1. The PlasMapper entry page provides a graphical user interface that allows users to upload a sequence from their local computer or paste a FASTA-formatted sequence into an input box for plasmid map generation. This page also includes many options that enable the user to customize the output.
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The feature identification and image rendering portion of PlasMapper
consists of four separate programs: BLAST, FIND-SITE, FORMAT
and CGView. BLAST (
9) is used to identify portions of the supplied
sequence that match promoters, terminators, selectable markers,
reporter genes and replication origins stored in the FSD. The
BLAST program parameters have been optimized for PlasMapper
by testing more than 50 randomly selected commercial plasmids
to ensure that the resulting annotations completely matched
those reported by the vendors. The FIND-SITE program, which
uses several components of BioJava (
http://www.biojava.org/),
is used to identify open reading frames (ORFs) and type II restriction
enzyme cutting sites. The third program (FORMAT), also written
in Java, generates formatted text output which displays the
plasmid sequence (60 bases per line, numbered, courier font)
with the requested annotations displayed in stacked, non-overlapping
positions above each sequence line. The final program (CGView)
uses the Java2D API to convert the results obtained from BLAST
and FIND-SITE into a graphical map. Specifically, CGView accepts
sequence feature information (feature name, feature type, position
and strand) and generates a collection of two-dimensional objects.
Each object's shape (an arrow or an arc), color, opacity and
position are adjusted according to the attributes of the feature
represented. After the objects are drawn, the feature names
are placed on the map using an iterative collision detection
and shifting process that results in a visually pleasing label
arrangement and no label overlap. Java classes included as part
of the Java API are used to convert the map into JPG and PNG
images. SVG output is generated using the Batik SVG Toolkit
(
http://xml.apache.org/batik/). Because SVG is a vector format,
SVG images can be scaled without any noticeable degradation.
Most web browsers can display SVG images using the freely available
Adobe SVG Viewer plug-in (
http://www.adobe.com/).
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PROGRAM OUTPUT
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PlasMapper generates both text and graphics output (JPG, PNG
or SVG format). The default view is the graphic image, with
a button to create the text view in another window.
Figure 2 provides an example of the range of text and graphics outputs
that can be produced. PlasMapper is designed to be interactive,
allowing users to readily go back and forth between the PlasMapper
home page and the output, so that new images or text can be
generated using a variety of display options. Using radio buttons,
text boxes and pull-down menus, users can control a large number
of display variables from the ‘Options’ portion
of the PlasMapper home page. Figure legends, color schemes,
shading, directional arrows, restriction enzyme labels and plasmid
feature labels (promoters, terminators, replication origins,
tags, etc.) may be easily turned on or off using radio buttons.
Similarly, custom feature titles, minimum ORF lengths, and image
titles may be added or edited using text boxes. Image formats,
image sizes, plasmid circle widths and gene arc widths may be
altered using pull-down list boxes. The images and textual output
generated from PlasMapper can be saved or copied directly to
the user's hard disk or placed into standard word-processing,
presentation or image manipulation programs. Images and text
generated by PlasMapper are kept in a /temp file and are stored
for not more than 24 h on the PlasMapper server. Similarly,
PlasMapper sessions that are inactive for more than 20 min are
terminated.
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Figure 2. A screenshot montage of PlasMapper output showing an example of two graphical views and a text view. The graphical views show labeled features including replication origins, restriction enzyme sites, selectable markers and reporter genes. The text view shows the individual DNA base pairs as well as the feature names.
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DISCUSSION
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The PlasMapper server provides a convenient and easily accessible
solution to plasmid annotation and drawing for users who normally
depend on freeware or free web servers such as EMBOSS (
10) or
The Sequence Manipulation Suite (
11). The simplicity and accessibility
of PlasMapper should also make it a useful tool for teaching
or training high school and university students in introductory
molecular biology or genetics courses. Furthermore, we believe
that the use of web-server technology should enable or encourage
the sharing of plasmid data and images among geographically
distant labs or between labs that normally use incompatible
software and/or computer platforms.
In trying to make the PlasMapper interface as simple as possible, some sacrifices in flexibility had to be made. No doubt some users may not want a specific label displayed or will dislike the default color scheme. Others may find certain rare restriction enzymes or gene features missing from the FSD. Likewise, the limited choice of plasmid circle or gene/marker widths may seem too restrictive. In many cases, these problems can be addressed by simply pasting the PlasMapper image into an image manipulation package to change the offending feature. The SVG format is helpful in this regard, since individual features and labels can be repositioned or modified using vector-graphics-capable software. For more specialized maps and annotations, commercial sequence analysis packages may be a more appropriate choice.
In summary, PlasMapper is a web server that permits the automated annotation and rendering of circular plasmids for both eukaryotic and prokaryotic vectors. It combines database-searching and pattern-matching techniques with a unique collection of plasmid-feature sequences to automatically generate publication-quality text and images. PlasMapper supports a wide range of display and formatting options and should make plasmid analysis and manipulation much simpler and far more accessible. The PlasMapper web server is freely accessible at http://wishart.biology.ualberta.ca/PlasMapper.
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Notes
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The online version of this article has been published under
an open access model. Users are entitled to use, reproduce,
disseminate, or display the open access version of this article
provided that: the original authorship is properly and fully
attributed; the Journal and Oxford University Press are attributed
as the original place of publication with the correct citation
details given; if an article is subsequently reproduced or disseminated
not in its entirety but only in part or as a derivative work
this must be clearly indicated.
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