PrimerDesigner.pm
PrimerDesigner is a GBrowse plugin, written by
Sheldon McKay, that uses use the
primer3 program to design PCR primers for selected
regions within the genome browser. Please feel free to contact the author or The Gbrowse mailing list for
help or more information.
The video below demonstrates designing PCR primers using the
PrimerDesigner plugin.
Contents
How to use the PrimerDesigner plugin
Accessing the plugin
- From GBrowse main page, the PrimerDesigner plugin, as well as other
installed plugins, can be accessed via the ‘Reports and Analysis’
menu.
- In GBrowse, navigate to the genomic region you are interested in, then
select ‘Design PCR primers’ from the menu and click ‘Configure’
- You will then see a view similar to this:
- At the top right corner is a navigation menu that allows you to zoom
and pan the displayed segment in a manner similar to the main Gbrowse
user interface.
Selecting the target for the PCR primers
- If no target region is specified, primers will be designed to amplify
the center of the displayed sequence, indicated by a vertical red
line.
- You can specify the target region for PCR primers using rubber-band
selection.
- To activate this feature, point your mouse on the scale bar at the top
of the image and click your mouse-button down, without releasing
it.
- Then drag your mouse to the left or right to highlight the region or
annotations you are interested in.
- In the example below, the first exon of gene F29C4.2 is highlighted
with rubber-band selection.
- Once you are done selecting, release the mouse button and the page
will reload with the selected region targeted for PCR primers
PCR primer design parameters
- Design parameters, such as primer length, GC content, etc can be
configured at the bottom of the page.
- Each parameter can be clicked for detailed information.
- Reasonable default options are provided for each parameter. These
options usually work but can be adjusted as desired.
- The size of the PCR product is calculated based on the size of the
selected region. This can be manually adjusted, if desired.
Predicted PCR Primer Results
- Once you have clicked ‘Design Primers’, you will see a results page
similar to the example below.
- The PCR primers are mapped back onto the image, so you can see the
location of the predicted PCR amplicon.
- Below the image, primer statistics are show.
- You can also click to expand the sections marked with the [+] icon
for more detailed reports.
Note On Quality Scores
- Occasionally you will see quality scores flagged in red.
- Optimal primer scores should be less than 1
- Higher scores often result from minor departures from optimality
criteria such as primer length, GC content, etc.
- If the primers do not fall to far outside of the requested parameters,
it is not serious.
- However, being mindful that the primers are only as good as the
available sequence will allow, making changes to the allowed product
size range or target coordinates will permit more optimal primers to
be selected.
How to install the PrimerDesigner plugin
First of all, you will need to
install GBrowse, with all of its prerequisites, such
as BioPerl and
Bio::Graphics
Dependencies
Primer3
- Follow this link to go the primer website at Sourceforge.
- Primer3 is a binary executable, developed by Steve Rozen and
colleagues, that the PrimerDesigner plugin uses to to the actual work
of designing primers
- The plugin passes the DNA sequence and primer design parameters to
this program and then parses the results
Installing Primer3
- Download or compile the
suitable primer3 binary executable for your website.
- If necessary, change the name of the binary to ‘primer3’ so that it is
recognized by the PrimerDesigner plugin
- The default path for primer is in ‘/usr/local/bin’. If you have
installed it elsewhere, be sure to the the ‘binpath’ option (see
below) in your configuration file.
Bio::PrimerDesigner
- Follow this link for information
about the Bio::PrimerDesigner package.
- Bio::PrimerDesigner is a set of perl modules that provide a low-level
API for the primer3 program.
- Note: Bio::PrimerDesigner is not part of BioPerl or GBrowse; you
will have to install this package for the Primerdesigner plugin to
work.
Installing Bio::PrimerDesigner
- Bio::PrimerDesigner is on CPAN (the Comprehensive Perl
Archive Network).
- To install it, you can use the
CPAN shell.
Configuration
The Configuration File
Activating the Plugin
To activate the Primer Designer plugin, simple add its name to the list
of available plugins in the configuration file for you data source.
For example:
# Installed plugins
plugins = Spectrogram SequenceDumper PrimerDesigner
NOTE: For this plugin to work properly, you must ensure that all
dependencies (described above) are that the GBrowse database has DNA
sequence.
Configurable Options
To configure the plugin, add a stanza like this at the end of your
[GENERAL] section:
[PrimerDesigner:plugin]
binpath = /usr/bin/
method = local
ispcr = http://genome.ucsc.edu/cgi-bin/hgPcr?db=cb3
binpath
The default value is ‘/usr/local/bin’
Use this option to specify where the primer3 binary is located, if not
in the default ‘/usr/local/bin’
If you are using a remote URL (primer3 is installed on another server),
specify a URL instead.
method
The default value is ‘local’
Recognized options are ‘local’ (primer3 installed on the same server as
Gbrowse) or remote (primer3 is installed on another server).
The ‘remote’ method is implicit if the binpath option is set to a URL
beginning with
‘http://’
default size
The default value is 10_000
This option specifies the default segment size to show in the
PrimerDesigner image
max range
The default value is 300
This option species the degree to which the PCR product size can depart
from the optimal size, as determined by the size of the selected target
area for PCR primers.
ispcr
This option can be set to a URL that point to the UCSC in silico PCR
sight.
If selected, in silico PCR results will be reported in the output of
this plugin
These results help to determine if the correct amplicon is found and
also indicate whether other PCR products
are amplified by the primer pair.
You must check the site to see if there is a genome sequence for you
species and modify the above example accordingly
PrimerDesigner and GBrowse 2
The PrimerDesigner plugin does not work with
GBrowse 2.00. Work is currently underway to fix
this before the next release of GBrowse.
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