JCVI Primer
Designer
Polymerase
chain reaction (PCR) is used in directed sequencing for the discovery of novel
polymorphisms. As the first step in PCR
directed sequencing, effective PCR primer design is crucial for obtaining high-quality
sequence data for target regions. Since
current computational primer design tools are not fully tuned with stable
underlying laboratory protocols, researchers may still be forced to iteratively
optimize protocols for failed amplifications after the primers have been
ordered. Furthermore, potentially
identifiable factors which contribute to PCR failures have yet to be
elucidated. This inefficient approach to
primer design is further intensified in a high-throughput laboratory, where
hundreds of genes may be targeted in one experiment.
We
have developed a fully integrated computational PCR primer design pipeline that
plays a key role in our high-throughput directed sequencing pipeline. Investigators may specify target regions
defined through a rich set of descriptors, such as Ensembl
accessions and arbitrary genomic coordinates.
Primer pairs are then selected computationally to produce a minimal amplicon set capable of tiling across the specified target
regions. As part of the tiling process,
primer pairs are computationally screened to meet the criteria for success with
one of two PCR amplification protocols.
In the process of improving our sequencing success rate, which currently
exceeds 95% for exons, we have discovered novel and
accurate computational methods capable of identifying primers that may lead to
PCR failures.
The
high-throughput PCR primer design pipeline has been very successful in
providing the basis for high-quality directed sequencing results and for
minimizing costs associated with labor and reprocessing. The modular architecture of the primer design
software has made it possible to readily integrate additional primer critique
tests based on iterative feedback from the laboratory. As a result, the primer design software,
coupled with the laboratory protocols, serves as a powerful tool for low and
high-throughput primer design to enable successful directed sequencing.
How to acquire the JCVI
Primer Design software.
How to install the JCVI
Primer Design Software.
Examples
How to run through a few key workflows.
Frequently Asked Questions
Answers to questions posed by users that
might be asked again.
Get a copy of the JCVI Primer Design
Manuscript
Kelvin Li, Anushka Brownley, Timothy B Stockwell, Karen Beeson, Tina C
McIntosh, Dana Busam, Steve Ferriera, Sean Murphy, Samuel Levy, “Novel computational
methods for increasing PCR primer design effectiveness in directed sequencing”,
BMC Bioinformatics 2008, 9:191 (11
April 2008)