**************   PickPrimer_VariantGenomic    ************************************************
  Pick Primer VariantGenomic program should 'be able' to build primers for the PCR reaction.
  The primers will be built externally to a genomic region, selectable by the user through chromosome, position and width (see the chart below)
  They will also be built in areas without known polymorphisms, derived from dbSNPs, GnomAD, etc. The user can select them based on a threshold of their frequency
  The user can add to the known polymorphisms, even of their own stored in a VCF format file.
 
   The application uses the main PickPrimer_ThermoHybrid program (self-made and present in this same platform) to analyze the sequence template and find the
  'best' primer to amplify the given region.

  The main program calculates the thermodynamics parameter (melting temperature, Entropy and Enthalpy variation, with the Nearest-Neighbor method.
  It uses data from SantaLucia et al.(1998) and for salt correction from von Ahsen et al. (2001).
  In case of multi-mismatch or terminal mismatches it use Mfold parameter by Zuker M. (2003) and made approssimation parameter.

  In addition to the positioning data of the region to be amplified, the user can enter the desired annealing temperature, the reagent concentrations,
  the optimal length, the type of polymerase used (hotStart, proofreading), etc.
  If desired, the user can force the program to use its own primers, in this way it is possible to evaluate the goodness of their primers.

  The primers proposed by the 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.



 
   The program extracts the genomic sequence (already marked by the variants) of a genomic region from the chromosome set and centered on the start and end positions,
      of a length that allows to obtain the maximum amplified in both parts of the region 
      The marking of variants prevents the construction of primers in regions where such variants exist.
 



                                around free              around free                      
                                  |.......|              |......|                
               p=polymorphism          Pos_start      Pos_end                     p=polymorphism
  --SEQ: --------p-p--------p-----[-------(--------------)------]---------------p----------p-pp------------
                                  [ -      free  region      -  ]       
                 -->       -->                                                 <--         <--   NO primer in these regione

                      -->                                             <--   good primer
                    prime for                                   primer rev
                        |- - - - - - - - amplicon - - - - -- - - - -  |


  The original PickPrimerThermoHybrid program controls hairpin-loop formation at the ends of amplicons that can prevent primer hybridization,
   but here, this function is not used because the template sequence is masked with variants that can prevent the analysis of any hairpin-loops.
   For the future, the implementation of the analysis of the ends of the amplicon is planned, but limited to the standard sequence.
  
  It is possible to insert a file containing particular variants, in VCF format, in order to prevent the construction of primers in regions where these variants are present.

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