UniPrime v2.10 - A workflow-based platform for improved Universal Primer design

September 2012

UniPrime is an open-source software which automatically designs large sets of universal primers by simply inputting a GeneID reference. UniPrime automatically retrieves and aligns orthologous sequences from GenBank, identifies regions of conservation within the alignment and generates suitable primers that can amplify variable genomic regions. UniPrime differs from previous automatic primer design programs in that all steps of primer design are automated, saved and are phylogenetically limited. We have experimentally verified the efficiency and success of this program. UniPrime is an experimentally validated, fully automated program that generates successful cross-species primers that take into account the biological aspects of the PCR.

The current version is UniPrime [2.10] (Ootoowerg / September 7th, 2012).

Text files associated with the UniPrime: README TUTORIAL INSTALL LICENSE.

The Linux/Unix code is publicly available.

Before installing UniPrime, your system must be compliant with the following requirements:

• Perl 5.005 or later.
• NCBI Blast+ package: UniPrime uses functionality provided by Blastn.
• Primer3 is mandatory since UniPrime is based on Primer3.
• T-coffee or GramAlign is mandatory if you plan to use multi-alignment option.
• PostgreSQL is mandatory since UniPrime store all the sequences, alignments and primers in a database.

Once you download, uncompress (bunzip2), and un-tar (tar xf), see the file INSTALL for the installation instructions.

Here is a simple example of a full execution of UniPrime. Each step can be reviewed with the web-companion interface. For a complete explanation consult the file README.

• Added a new locus and use the human OAZ1 gene as prototype. The GeneID (4946) is retrieved from GenBank (from the command line in a Terminal window):

./uniprime.pl --action=locus --target=4946 -v

or

./uniprime.pl --action=locus --target=mysequence.fasta -v

• Use the locusID generated at the previous step (e.g. L3.1) and look for orthologues within the class Mammalia and an e-value of 2e^-50 as threshold:

./uniprime.pl --action=ortho --target=L3.1 -e=2e-50 --query=Mammalia[ORGN] -v

• Use the locusID (e.g. L3.1) and establish an alignment and a consensus sequence with a threshold of 60%:

./uniprime.pl --action=align --target=L3.1 -c=60 -v

• Use the AlignmentID (e.g. A3.2) and design the primer set:

./uniprime.pl --action=primer --target=A3.2 -v

• Optionally, use the primer set (e.g. X3.3) and do a virual PCR within the Class Mammalia of the WGS databank of the NCBI.

./uniprime.pl --action=vpcr --target=X3.3 --db=wgs --query=Mammalia[ORGN] -v