Locate exon-exon junctions

Introns are identified according to annotations related to the NCBI RefSeq transcript id.

Find UPL probe

UPL probes are mapped to the transcript sequence. For human data, each target site is verified for known SNP with a frequency higher than 1% using dbSNPv155.

Find PCR primers

For each target site, we use Primer3 to find the 5 best pair of primers. BLAST is then used on every pair of primers against the transcriptome to ensure primer uniqueness. As the amount of Blast hit is used to rank an assay, hits on various isoform of a same gene are merged to a single hit. For a given pair of primers, if there is no hit on the transcriptome, BLAST is used again on the genome.
Primers with a SNP of high frequency (above 1%) overlapping one of the last 3 bases of the primer sequence are discarded in order to avoid weak primer binding.

Score and rank each assay to promote

• Assays without known SNP overlapping the UPL probe binding site.
• Assays without cross hybridations to other genomic regions (BLAST result).
• Robust design of primer pairs by using Primer3 penalty score and minimizing melting temperature variation between the primers.

Present results

Based on their global score, the best pair out of the 5 primer pairs for each target site is conserved.
The assay details from each target site can be accessed by the user when clicking on the probe ID of the assay table. These details include the sequence, length, melting temperature and GC content of each primer as well as the full amplicon sequence.