# Turn-back primer optimization computer scripts
# Copyright: (c) 2011 RIKEN Omics Science Center
# Author: Yasumasa Kimura (y.kimura.g@gmail.com)
# Version: 1.0

0. Prepare softwares and files 

	* Softwares below must be installed before running our scripts : 
		ruby (http://www.ruby-lang.org/en/)
		R (http://www.r-project.org/)
		ssearch35_t (included in fasta-35 ftp://ftp.ebi.ac.uk/pub/software/unix/fasta/fasta3/)
		UNAfold package (unafold-3.6 http://dinamelt.bioinfo.rpi.edu/download.php)
		FastaGrep (http://bioinfo.ebc.ee/download/)

	* File needed : 
		Script : 
			TP_loop.rb
			DNA_property.rb
			P.calculate.fit.y.max.R
			P.calculate.parameter.rb
			P.get.TP_sequence.3end.rb
			parameter.R

		Files of the fitted model : 
			r134_04_lasso.neg.paper.max.beta.pval.txt
			r134_04_lasso.neg.paper.max.scale.txt
			r134_04_lasso.pos.paper.max.beta.pval.txt
			r134_04_lasso.pos.paper.max.scale.txt


1. Make target file containing your target sequence ( described as <target file> in later explanation )


2. Make input configuration file for producing primer sequences ( described as <input file> in later explanation )

	file format : 
		target_file    (target sequence file name, made in 1.)
		wt_mt          0 (please ignore this parameter)
		name           (name of the target)
		annl_3end      (3'end positions of annealing site on the target sequence, separated by comma ",")
		annl_5end      (limit of 5'end position)
		tb_3end        (3'end positions of turn-back site on the target sequence, separated by comma ",")
		tb_5end        (limit of 5'end position)
		l_annl         (lengths of anneal region (X), separated by comma ",")
		l_tb           (lengths of turn-back region (Z), separated by comma ",")
		y              (lengths between anneal and turn-back (Y), separated by comma ",")
		direction      (direction of primers "F" or "R")
		base_pos       0 (position taken as an original point in the target sequence)
		n_group        (the number of output files divided for parallel computing)

	example (forward, "i.input.example.f.txt") : 
		target_file    i.target.example.txt
		wt_mt          0
		name           example
		annl_3end      128,129,130,131,132,133,134,135,136,137,138,139,140
		annl_5end      100
		tb_3end        158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180
		tb_5end        130
		l_annl         16,18,20,22
		l_tb           8,10,12,14
		y              15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35
		direction      F
		base_pos       0
		n_group        1

	example (reverse, "i.input.example.r.txt") : 
		target_file    i.target.example.txt
		wt_mt          0
		name           example
		annl_3end      237,241,245,249
		annl_5end      300
		tb_3end        195,201,207,213
		tb_5end        250
		l_annl         18,22
		l_tb           10,14
		y              15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35
		direction      R
		base_pos       0
		n_group        1


3. Run "P.get.TP_sequence.3end.rb" to generate primer sequences

	ruby P.get.TP_sequence.3end.rb  <input file>  

		* <primer file> will be generated as an output file.
		The file name is "r.TP_sequence.(target name).(primer type).(base_pos).txt".
		This file is used in 4. as a input file.


4. Run "P.calculate.parameter.rb" to calculate parameters for each primer

	ruby P.calculate.parameter.rb  <primer file>  <target file>  <parameter file>  <hetero primer>
	
		* <parameter file> will be generated as an output file. You have to put the name.
		  This file is used in 5. as a input file.

		* <hetero primer> file is optional. The file is tab-delimited format :
		  1. primer id
		  2. primer sequence
		  if this file is specified then the parameters related to hetero primers will be calculated.


5. Run "P.calculate.fit.y.max.R" to predict reaction speeds

	R  --vanilla  --slave  --args  <parameter file>  <pred target speed file>  <pred background speed file>  <  P.calculate.fit.y.max.R 

		* The following two files will be generated as output files. You have to put the names.
		  - <pred target speed file> : the file contains predicted target reaction speeds.
		  - <pred background speed file> : the file contains predicted background reaction speeds.
		  Those predicted reaction speed file are tab-delimited format :
		  1. primer id
		  2. primer sequence
		  3. predicted reaction speed

Example:

	i. ruby P.get.TP_sequence.3end.rb  i.input.example.r.txt  

	ii. ruby P.calculate.parameter.rb  r.TP_sequence.example.Tr.0.txt  i.target.example.txt  r.parameter.example.txt  i.hetero.example.f.txt

	iii. R  --vanilla  --slave  --args  r.parameter.example.txt  r.speed.target.example.txt  r.speed.bckgrd.example.txt  <  P.calculate.fit.y.max.R 

	iv. The result will be "r.speed.target.example.txt" and "r.speed.bckgrd.example.txt" for the target and background amplification speed, respectively 


# Reference: 
Kimura,Y., de Hoon,M.J.L., Aoki,S., Ishizu,Y., Kawai,Y., Kogo,Y., Daub,C.O., Lezhava,A., Arner,E. and Hayashizaki,Y. (2011) 
Optimization of turn-back primers in isothermal amplification. Nucleic acids research, 39, e59.