Batch bisulfite primer design tool version 2 (NEW : Multiple results per template, allowed to set number of C's within a primer)

mp_primer_v2.sh

#!/bin/bash

printf "\n *** BIS BATCH PRIMER version 2.0 ***"
printf "\n\n !!! 'Primer3 & fastx-toolkit' must be installed on the system.\n\n !!! Edit parameters (e.g. sizes, Tm, and etc) before start\n\n "
printf "\n\n  Usage : \n    ./mp_primer.sh FASTA PARAMETER \n\n"
printf "   >>>  input FASTA = "$1
printf " \n   >>>  parameters  = "$2
printf "\n\n\n  ()()() Running... \n\n"

if [ -f $1 -a -f $2 ]; then
		mkdir tmp
		cd tmp

		cp ../$2 .
		cat ../$1 | while read L; do echo $L"_lower";read L ;echo "$L" | rev | tr "ATCG" "TAGC" ; done > $1.rev  # reverse_complement
		cat ../$1 $1.rev > $1.comb

		sed 's/ /_/g' $1.comb | fasta_formatter -t | awk '{print $1"\t"toupper($2)}' |sed 's/CG/NG/g' | sed 's/C/T/g' > $1.comb.tab
		# mask 'CG' to 'NG' & covert rest 'C' to 'T'

		cut -f 1 $1.comb.tab | sort | while read line; do grep -w $line $1.comb.tab > $line.split; done
		# split each header into a single file

####################################
# sequence file generator (Add titles to tab. "SEQUENCE_ID=", "SEQUENCE_TEMPLATE=")
		for s in ./*.split
		do
			awk '{print "SEQUENCE_ID="$1,"\n","SEQUENCE_TEMPLATE="$2}' $s | sed 's/ //g' > $s.seq
		done

####################################
# parameter file generator
		for s in ./*.split           # to generate a list of target sequence (CpG) positions
		do
			cut -f 2 $s | grep -bo 'NG' | sed 's/:/\t/g' | cut -f 1 | awk '{print $1+1",2"}' | tr '\n' ' ' | \
			awk '{print "SEQUENCE_TARGET="$0}' | cat - $2 > $s.param
		done

####################################
# combine sequence & parameter files
		cut -f 1 $1.comb.tab | uniq | while read line; do cat $line.split.seq $line.split.param > $line.p3in ; done

####################################
# PRIMER3
		for i in ./*.p3in          # run PRIMER3
		do
			primer3_core $i > $i.p3out
		done

####################################
# print ouf PRIMER3 results
		for r in ./*.p3out
		do
			grep 'SEQUENCE=\|LEFT_.=\|RIGHT_.=\|LEFT_._TM\|RIGHT_._TM' $r | sed 's/=/\t/g' | cut -f 2 | paste -d "\t" - - - - - - | sed 's/,/\t/g' | \
			perl -ne 'print if !/N/ || /\b[ACTG]{0,4}N/' | \
			awk 'BEGIN{FS=OFS="\t"}{gsub("N","Y",$1);gsub("N","R",$2);print $1,$2,$5-$3+1,$3,$5,$4,$6,$7,$8}' > $r.int
		done

		for i in ./*.int
                do
                        awk '{print FILENAME"_"NR,$0}' $i | sed 's/ /\t/g;s/.p3in.p3out.int//g' | awk -F "/" '{print $2}'
                done | sort -k1 > $1.p3outs
		echo 'ID	Forward	Reverse	Product_Size	Product_Start	Product_End	Len_forward	Len_reverse	Tm_forward	Tm_reverse' | \
		cat - $1.p3outs > ../$1.p3.table

# p3out for isPCR
		for r in ./*.p3out
		do
			grep 'SEQUENCE=' $r | paste -d "\t" - - | sed 's/=/\t/g' | cut -f 2,4| perl -ne 'print if !/N/ || /\b\S{0,4}N/' | \
			awk '{gsub("N","T",$1);gsub("N","A",$2); print}' > $r.ispcr
		done

	        for i in ./*.ispcr
		do
			awk -F "/" '{print FILENAME"_"NR,$0}' $i | sed 's/ /\t/g;s/.p3in.p3out.ispcr//g' | awk -F "/" '{print $2}'
		done | sort -k1 > ../$1.isPcrInput

#################################
# primer design report
		printf "\n\n   *** PRIMER REPORT\n\n   *  INPUT HEADERS  =  "
		grep -c ">" ../$1
		printf "\n   *  DESIGNED PRIMER PAIRS  =   "
		cat ../$1.isPcrInput | wc -l
		printf "\n   *  PASSED HEADERS = "
		sed 's/_/\t/g' ../$1.isPcrInput | cut -f1 | uniq | wc -l
		printf "\n   *  FAILED HEADERS  =  "
		sed 's/_/\t/g' ../$1.isPcrInput | cut -f1 | sort | uniq > header.isPcrInput
		grep ">" ../$1 | sed 's/>//g'  | sort | uniq > header.Input
		join --check-order -t: -a 1 -o 1.1 2.1 -1 1 -2 1 header.Input header.isPcrInput |\
		awk -F':' '{if($2 == "") {print $1}}' | tr '\n' ' '

                printf "\n\n   >>> '$1.p3.table' has been generated! (Use this file to order primers)\n"
                printf "   >>> '$1.isPcrInput' has been generated! (Use this file to obtain amplicon sequences)"
		printf "\n\n   !!!  Done  !!!\n\n"

                cd ..
#                rm -r tmp/


else
	printf "\n\n   !!! ERROR:: FASTA or parameter file is missing!\n\n"
fi