Optimus Primer is managed by the Génome Québec & Montreal Heart Institute Pharmacogenomics Centre.

Optimus Primer Help

Input Files

There are two types of input files that can be uploaded into the pipeline.

  1. Gene or Feature File
  2. Configuration File (optional)

Region Variables

The region variable sections allows you to define global parameters for the run and upload user input

Run Name
A unique name given to the particular run of the pipeline. This name will be appended to output files
Gap Size
The length, in base-pairs, for the maximum distance between exons before they are grouped into a single region for amplification
Desired Amplicon Size
The length, in base-pairs, of the optimal amplicon size. Exons that are larger then this size will be divided into multiple amplicons
Added Sequence
The amount of sequence to be added upstream and downstream of each exon for primers to be designed within.
Minimum Overlap of Amplicons
The minimum amount of sequence in base-pairs to overlap between adjacent amplicons in exons that require more than one amplicon to cover
Input File Type
The type of file to be uploaded to define regions of interest. Either Gene List of Feature file (See Input Files)
Configuration Type
The method used to define Primer3 design parameters, either manually or by file upload

Primer3 Variables

In this section, the parameters used for primer design can be defined. Parameters for four successive iterations of Primer3 can be defined. Amplicons that fail the first pass of primer design are automatically passed on to the next iteration. Primer3 source code can be foundhere The development of Primer3 and the Primer3 web site was funded by Howard Hughes Medical Institute and by the National Institutes of Health, National Human Genome Research Institute. under grants R01-HG00257 (to David C. Page) and P50-HG00098 (to Eric S. Lander). Full documetation is available here. Selected definitions are described below.

Product Size
Minimum, Optimum, and Maximum lengths (in bases) of the PCR product. Primer3 will not generate primers with products shorter than Min or longer than Max, and with default arguments Primer3 will attempt to pick primers producing products close to the Optimum length.
Primer Tm
Minimum, Optimum, and Maximum melting temperatures (Celsius) for a primer oligo. Primer3 will not pick oligos with temperatures smaller than Min or larger than Max, and with default conditions will try to pick primers with melting temperatures close to Opt.

By default Primer3 uses the oligo melting temperature formula and the table of thermodynamic parameters given in Breslauer et al. 1986, DOI:10.1073/pnas.83.11.3746 For more information see caption Table of thermodynamic parameters

Primer GC%
Minimum, Optimum, and Maximum percentage of Gs and Cs in any primer or oligo.
CG Clamp
If selected,requires that primers have either a G or C at the 3' end of both the left and right primer.
Number To Return
The maximum number of primer pairs to return. Primer pairs returned are sorted by their "quality", in other words by the value of the objective function (where a lower number indicates a better primer pair). Note: Maximum number return for Optimus Primer is 5.
Product Size Range
A list of product size ranges, for example
    150-250 100-300 301-400
Primer3 first tries to pick primers in the first range. If that is not possible, it goes to the next range and tries again. It continues in this way until it has either picked all necessary primers or until there are no more ranges.
Lowercase masking
If checked candidate primers having lowercase letter exactly at 3' end are rejected. This option allows to design primers overlapping lowercase-masked regions. This property relies on the assumption that masked features (e.g. repeats) can partly overlap primer, but they cannot overlap the 3'-end of the primer. In other words, the lowercase letters in other positions are accepted, assuming that the masked features do not influence the primer performance if they do not overlap the 3'-end of primer.
SNP masking
If checked, known SNPs from dbSNP build 130 will be masked as "Ns" in sequenced submitted to Primer3. Optimus Primer will not allow primers containing "Ns" to be designed.

Output Files

In the zip file that is produced at the end of your run (see the sample output page for an example) you will find the following files:

Final_Table_run-name.xls
Contains the best isPCR validated primer pair for each amplicons."No isPCR validated primers" indicates that primers were designed for this amplicon however they did not pass isPCR.
amplicons_with_no_primers.txt
Contains all amplicons for which no possible primer pair was designed under specified parameters
gene_coverage_report.txt
Summary of % coverage per exon and gene for all isPCR validated primer pairs (Only included if a gene list is submitted)
genes_no_exons_run-name.txt
List of genes not found in RefSeq database
regions_details.txt
Gene name, chromosome, starting and ending position for each exon identified. As well lists how exons in close proximity to each other are grouped together.
PCR_run-nam_BED.out
isPCR output file in BED format for all primer pairs designed. Can be uploded as a custom track on the UCSC genome browser to visualize isPCR validated amplicons.
genelist.txt
Submitted gene list (Only included if a gene list is submitted)
All_Designs_Table_run-name.csv
Every primer pair that was designed for every amplicon in all iterations
file_run-name.conf
Configuration file containing Region and Primer3 parameters used for run. Note: Can be uploaded to OP for future runs
pipeline_input_run-name.txt
Feature file for all amplicons Note: can be uploaded to OP for addition runs