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== NGS_GenomeMarkerDesigner                                                  ==
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INTRODUCTION:
=============

NGS_GenomeMarkerDesigner is a perl pipeline to design markers based in a 
reference sequence (for example a genome sequence) and a .vcf file created
after call SNPs from a bam/sam mapping file.

For now it will be able to create two types of markers:

   a) SNP marker based in the primer design using the SNP nucleotide

      Example:
         >>>>>>>>>>>>>>*
      5'-ATCGTGTCGACGATGGCAGGATGACGACGGATGAGCACAGGGGCATAGACAGGACAGACGACAGT-3'
      5'-..............C..................................................-3'
                                                           <<<<<<<<<<<<<<<

      15 Kmer primers for 75 bp insert
      Fw => 5'-ATCGTGTCGACGATG-3'
      Rv => 5'-ACTGTCGTCTGTCCT-3'
       
   b) CAPS markers

      Example:
                                     -!----
      5'-ATCGTGTCGACGATGGCAGGATGACGACGGATCCGCACAGGGGCATAGACAGGACAGACGACAGT-3'
      5'-..............................C..................................-3

      15 Kmer primers for 75 bp insert, CAPS marker for 30 and 45 bp
      Fw => 5'-ATCGTGTCGACGATG-3'
      Rv => 5'-ACTGTCGTCTGTCCT-3'

   c) SSR markers (Under future development)

      Example:
                           |+||+||+||+||+||+||+| 
      5'-ATCGTGTCGACGATGGCAGGAGGAGGAGGAGGAGGAGGACGGGCATAGACAGGACAGACGACAGT-3'
      5'-.................................------..........................-3

      15 Kmer primers for 75 bp insert, SSR markers for 75 and 69 bp
      Fw => 5'-ATCGTGTCGACGATG-3'
      Rv => 5'-ACTGTCGTCTGTCCT-3'


PROCESS:
========

1) Restriction Enzyme Map of the Reference Sequence (SeqRef2RestrictMap).

   The reference sequence will be analyzed to create a restriction map
   stored as gff3 file

2) SNP filtering based in: QUAL (SNP call quality), AF (allelefrequency) and 
   DP (depth)) (SnpFilter) 

3) Calculation of how many restriction maps can be affected by SNP contained
   into the .vcf file (SnpXRestrictMap).

3) Output and marker generation (MarkerGenerator)



REQUERIMENTS:
=============

 + Perl v5.10.1
 + Perl Package: Bioperl v1.0069       (Sequence File Parsing/Restrict. Mapping)
 + Perl Module:  Config::General v2.48 (Configuration File Managing)
 + Perl Module:  R::YapRI v0.04        (Perl-R Interaction for Graphics)
 + R v2.13                             (R for graphics) 
 + Primer3                             (Primers Design)

USAGE:
======

1) As separate scripts:

   SeqRef2RestrictMap -r <refseq> 
                      -s <restriction_enzyme_sites_catalog>
                      -o <output_basename>

   SnpFilter          -v <vcf_file>
                      -d <min_depth>
                      -a <min_allele_freq>
                      -q <min_qualscore>

   SnpXRestricMap     -v <filtered_vcf_file>
                      -g <restriction_sites_map_gff3>
                      -o <output_basename>

   MarkerGenerator    -r <refseq>
                      -v <filtered_vcf_file>
                      -g <restriction_sites_with_SNPs_gff3>
                      -k <primer_kmer>
                      -s <pcr_product_size>
                      -m <markers_per_position>
                      -d <min_distance_between_markers>
                       
2) As a pipeline:

   GenomeMarkerDesigner -r <refseq>
                        -v <vcf_file>
                        -s <restriction_enzyme_catalog>
                        -o <output_dir> 
                        -c <configuration_file> [-V]

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Created by: Aubombarely, 2011-Dec-14