Dependencies:

1.	Perl models: Getopt::Long, Time::Local, and Cwd. Installation steps:
>cpan
cpan>install Getopt::Long
cpan>install Time::Local
cpan>install Cwd
2.	Primer3. See installation instruction on the Primer3 website (https://sourceforge.net/projects/primer3/). 
3.	BLASTN. See installation instruction on the NCBI website (http://www.ncbi.nlm.nih.gov/books/NBK279690/)
4.	R base and Bioconductor package “ggbio” (optional). See installation instruction in R website. 

Required files:

1.	--design <three design: individual, region, and SM>. Primer design strategy with three options: individual: only one primer pair in one region (one region per line in the input file); region: primer design in a moving window and step in one region; SM: small mutation, only one primer to cover the causal small mutation, such as small deletion or SNPs. 
2.	--input <input files with chromosome positions>. Input file contains the genomic coordinate, format: gene/exon_name	chrosomome_number	Start	End (GRMZM2G162508_E2	1	240842179	240843233). One gene/exon in one line, and multiple lines are accepted. 
3.	[--winstep <Window and step for region design>]. Set moving windows and step. Default: Default (500,200): window, 500bp; step, 200bp.
4.	[--smrange <DNA length for SM design strategy>]. DNA length for SM design strategy used to design forward and reverse primers before and after the small mutation. 
5.	--reference <genome reference fasta file>. Path to the fasta reference file.
6.	[--primer3 <primer3 parameters>]. Optional. Set up your own Primer3 parameters to design a pair of primers. See Primer3 manual for more details.
7.	--output|-o <output.prefix>. Path to output files' prefix. 


Example command line:
perl gppd.pl --design=region --input=example.txt --reference=maize.3_4.ref.fa --threads=20 --output=test

