Main default data: considering all variants, Temp.annealing is 56 ° C, optimal amplicon length is 500 pb, standard PCR solution. Genome data This section allows to set the genomic data and select a central region (core region). The primers will be built laterally to this region.. Mark the genomic variants This section allows to mark the genome with the variants (according to their frequency) in order to prevent the construction of the primes where these variants exist. Genomic polymorphism were derived from: - dbSNPS 151 (GRCh37 and GRCh38) from NCBI (file 00_All.vcf, only SNPs validate) - Frequencies from GnomAD: gnomAD v2.1.1 for GRCh37/hg19 (exomes and genomes) gnomAD v3.0 for GRCh38/hg38 (genomes)
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PCR reaction this section allows to select the parameters of the PCR reaction click on the suggested links to get explanations about the choices Primer this section allows to characterize the primers and/or choose their own click on the suggested links to get explanations about the choices Display this section allows to select the desired type of display (on screen or on file), the quantity of copies of primers to be listed and how many times the same primer must be used in combinations click on the suggested links to get explanations about the choices |
They will also be built in areas without known polymorphisms, derived from dbSNPs, GnomAD, 1000 genomes, etc. the user can select them based on frequency threshold.
Moreover, The user can add to the known polymorphisms, even of their own stored in a VCF format file.
The primers proposed by the program, analyzed with thermodynamic parameters with the Nearest-Neighbor method by our PinkPrimerThermoHybridPCR program, will have a length appropriate to the set temperature and will be analyzed for the content in bases, stability at 3 ', presence of any stretch of similar bases, possibility to form hairpin-loops and dimers (self and hetero).
The program assigns a penalty for each aspect analyzed and the primers with the minor penalty will be proposed.