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primer_evaluation.pl
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primer_evaluation.pl
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#!/usr/bin/perl -w
use strict;
use warnings;
use Getopt::Long;
use Data::Dumper;
use FindBin qw($Bin $Script);
use File::Basename qw(basename dirname);
require "$Bin/common.pm";
require "$Bin/product.pm";
require "$Bin/self_lib.pm";
require "$Bin/path.pm";
require "$Bin/io.pm";
my $BEGIN_TIME=time();
my $version="1.0.0";
#######################################################################################
# ------------------------------------------------------------------
# GetOptions
# ------------------------------------------------------------------
my ($foligo,$fbound,$fprobe,$fkey,$outdir);
our $REF_GRCh37;
my $fdatabases = $REF_GRCh37;
my $ptype = "face-to-face";
my $PCRsize=1000;
my $min_eff=0.00001;
my $min_tm_spec=45;
my $opt_tm = 60;
my $opt_tm_probe = 70;
my $max_prodn=50;
my $thread = 10;
my $etype="SinglePlex";
my ($AllEvalue, $OutAllProduct, $Methylation, $NoSpecificity);
my $range = "120,160,80,200";
GetOptions(
"help|?" =>\&USAGE,
"io:s"=>\$foligo,
"ib:s"=>\$fbound,
"id:s"=>\$fdatabases,
"k:s"=>\$fkey,
"tp:s"=>\$ptype,
"ep:s"=>\$etype,
"AllEvalue:s"=>\$AllEvalue,
"OutAllProduct:s"=>\$OutAllProduct,
"Methylation:s"=>\$Methylation,
"NoSpecificity:s"=>\$NoSpecificity,
"rd:s"=>\$range,
"tm:s"=>\$opt_tm,
"tmb:s"=>\$opt_tm_probe,
"sz:s"=>\$PCRsize,
"mp:s"=>\$max_prodn,
"stm:s"=>\$min_tm_spec,
"me:s"=>\$min_eff,
"td:s"=>\$thread,
"od:s"=>\$outdir,
) or &USAGE;
&USAGE unless ($foligo and $fkey);
$outdir||="./";
`mkdir $outdir` unless (-d $outdir);
$outdir=AbsolutePath("dir",$outdir);
my @lendif=(0,4,0,8); ## tm diff between F and R primer
my @tmdif=(0,3,0,6); ## tm diff between F and R primer
my @rdis=split /,/, $range;
my ($mind, $maxd)=($rdis[2], $rdis[3]);
my ($fulls_pos, $fulls_dis, $fulls_lend, $fulls_tmd, $fulls_prod)=(20,30,10,10,30);
my ($w1, $w2, $wr, $wp)=(0.3, 0.3, 0.4, 0);
my $fevalue;
my $ftype;
my $info = `tail -1 $foligo`;
chomp $info;
my $col = scalar(split /\s+/, $info);
my $idx;
if($col ==2){
$ftype="Common";
$idx = 0;
}elsif($col==24 || $col==23){
$ftype="Evalue";
$idx = 3;
$fevalue = $foligo;
}
if(!defined $fbound){
### evalue single oligo
if($ftype ne "Common"){
die "Wrong file type: must be Common(2column: id seq) when not defined -ib!\n";
}
my $ftemplate = "$outdir/$fkey.template.fa";
`less $foligo|perl -ne '\''{chomp; \@a=split; print \">\$a[0]\\n\$a[1]\\n\";}'\'' >$ftemplate`;
my $cmd = "perl $Bin/oligo_evaluation.pl --nohead -p $foligo -t $ftemplate -d $fdatabases -thread $thread -stm $min_tm_spec --NoFilter -k $fkey -maxtime 100000000 -od $outdir";
if(defined $Methylation){
$cmd .= " --Methylation";
}
if(defined $NoSpecificity){
$cmd .= " --NoSpecificity";
}
&Run($cmd);
$fevalue = "$outdir/$fkey.evaluation.out";
$fbound = "$outdir/$fkey.bound.info";
}else{
if($ftype ne "Evalue"){
die "Wrong file type: must be Evalue type(24column) when defined -ib!\n";
}
}
my %record;
my $is_probe=0;
open(I, $foligo) or die $!;
while(<I>){
chomp;
next if(/^$/ || /^#/);
my @unit=split /\s+/, $_;
my $id=$unit[$idx];
my ($tid, $type)=$id=~/(\S+)-([12FRP])/;
if($type eq "P"){
$is_probe=1;
}
$record{$id}=0;
}
close(I);
if($is_probe){
($fulls_pos, $fulls_dis, $fulls_lend, $fulls_tmd, $fulls_prod)=(40,20,10,10,20);
($w1, $w2, $wr, $wp)=(0.25, 0.25, 0.25, 0.25);
}
### evalue combination oligos
my %bound;
my %product;
my %productp;
my @all_alignments = ();
if(!defined $NoSpecificity){
my %boundm;
&SHOW_TIME("#Read in Bound file");
open(B, $fbound) or die $!;
while(<B>){
chomp;
my ($id, $strand, $chr, $pos5, $seq, $tm, $end3_base, $mvisual)=split /\t/, $_;
next if(!exists $record{$id});
$record{$id}=1;
my ($tid, $type)=$id=~/(\S+)-([12FRP])/;
if(!defined $tid || !defined $type){
die "Wrong oligo ID $id: Primer ID must end by -F/R/1/2, eg. xxx-F, xxx-R, xxx-1, xxx-2; Probe ID must end by -P, eg. xxx-P!\n";
}
my $len = length $seq;
my $pos3=$strand eq "+"? $pos5+$len-1: $pos5-$len+1;
my $a = [$chr, $strand, $pos3, $pos5, $tm, $end3_base, $mvisual, $tid, $type, undef, undef, undef, undef];
# no-use eff eff_tm eff_end
push @{$bound{$type}{$tid}}, $a;
push @all_alignments, $a;
}
close(B);
foreach my $id(keys %record){
if($record{$id}==0){
print "Warn: $id not exist bound info and cannot evalue its products!\n";
}
}
my @tps = sort {$a cmp $b} keys %bound;
&SHOW_TIME("#Evalue");
&caculate_product_evaluation(\@all_alignments, $ptype, \%product, \%record, $PCRsize, $opt_tm, $min_tm_spec, $mind, $maxd, $min_eff, @all_alignments*@all_alignments, $etype, $AllEvalue, $tps[0]);
if($is_probe==1){#Probe
foreach my $tid(keys %{$bound{"P"}}){
# probe num on products
&probe_bounds_on_products($tid."-P", $bound{"P"}{$tid}, $product{$tid}{$tid}, \%productp, $PCRsize, $opt_tm_probe, $min_tm_spec+8); #my ($id, $abound, $aprod, $aresult)=@_;
}
}
}
### output final.result
if($ftype eq "Common"){ ## score
open(I, $fevalue) or die $!;
my @ids;
my %info;
while(<I>){
chomp;
next if(/^$/ || /^#/);
my ($abase, $afeature, $ameth, $aspec, $bnumtm)=&read_evaluation_info($idx, $_, $Methylation, 1); #spec: ($bnum,$btm,$binfo)
my ($id, $seq, $len) =@{$abase}[$idx..($idx+2)];
my ($tid,$tp)=$id=~/(\S+)-([12FRP])$/;
$tp=~s/F/1/;
$tp=~s/R/2/;
@{$info{$tid}{$tp}}=($abase, $afeature, $ameth, $aspec, $bnumtm);
push @ids, $id;
}
close(I);
if(defined $OutAllProduct){
open(P, ">$outdir/$fkey.final.pair.product") or die $!;
}
open(O, ">$outdir/$fkey.final.result") or die $!;
print O "##ScoreOligo: total score | ". &score_des("Primer", $Methylation)."\n";
if($is_probe==1){
print O "##ScoreOligo(Probe) : total score | ". &score_des("Probe", $Methylation)."\n";
}
print O &final_evalue_head($Methylation, $NoSpecificity)."\n";
#### score
my %score_rel;
my %score_info;
my %prod_info;
foreach my $tid(sort {$a cmp $b} keys %info){
my ($abase1, $afeature1, $ameth1, $aspec1, $bnumtm1) = @{$info{$tid}{1}};
my ($abase2, $afeature2, $ameth2, $aspec2, $bnumtm2) = @{$info{$tid}{2}};
my ($id1, $seq1, $len1) =@{$abase1}[$idx..($idx+2)];
my ($id2, $seq2, $len2) =@{$abase2}[$idx..($idx+2)];
## score oligo
my ($tid,$tp1)=$id1=~/(\S+)-([12FRP])$/;
my ($s1, $s2, $score_info1, $score_info2);
if(defined $Methylation){
($s1, $score_info1)=&primer_meth_score($opt_tm, $len1, @{$afeature1}, @{$ameth1}, $bnumtm1);
($s2, $score_info2)=&primer_meth_score($opt_tm, $len2, @{$afeature2}, @{$ameth2}, $bnumtm2);
}else{
($s1, $score_info1)=&primer_oligo_score($opt_tm, $len1, @{$afeature1}, $bnumtm1);
($s2, $score_info2)=&primer_oligo_score($opt_tm, $len2, @{$afeature2}, $bnumtm2);
}
@{$score_info{$id1}}=($s1, $score_info1);
@{$score_info{$id2}}=($s2, $score_info2);
## score for relation
my ($sp, $score_infop)=(0, "");
my $spos = $fulls_pos;
if(exists $info{$tid}{"P"}){
my ($abase, $afeature, $ameth, $aspec, $bnumtm) = @{$info{$tid}{"P"}};
my ($id, $seq, $len) =@{$abase}[$idx..($idx+2)];
my ($is_G5, $CGd) = &G_content($seq);
if(defined $Methylation){
($sp, $score_infop)=&probe_meth_score($opt_tm_probe, $len, @{$afeature}, @{$ameth}, $bnumtm, $is_G5, $CGd);
}else{
($sp, $score_infop)=&probe_oligo_score($opt_tm_probe, $len, @{$afeature}, $bnumtm, $is_G5, $CGd);
}
@{$score_info{$id}}=($sp, $score_infop);
my ($pnum, $apeff, $apinfos)=&get_highest_bound($productp{$id}, 1000000, "Eff");
my $pos=$apinfos->[0]=~/^(\S+)\//;
$spos=int(&score_single($pos, $fulls_pos, (1, 5, 0, 10))+0.5);
my ($peffs, $pinfos);
if($pnum!~/\+/ && $pnum<=3){
$peffs = join(",", @{$apeff});
$pinfos = join(";", @{$apinfos});
}else{
$peffs = join(",", @{$apeff}[0..2]);
$pinfos = join(";", @{$apinfos}[0..2]);
}
@prod_info{$id}=($pnum, $peffs, $pinfos);
if(defined $OutAllProduct){
print P ">$id\t$pnum\n";
my @peff=@{$apeff};
my @pinfo=@{$apinfos};
for(my $i=0; $i<@{$apeff}; $i++){
print P join("\t", $apinfos->[$i], $apeff->[$i]),"\n";
}
}
}
# score for tm diff
my $tmdif=abs($afeature1->[0] - $afeature2->[0]);
#print "tmdif:\t", join("\t", $tmdif, $fulls_tmd, @tmdif),"\n";
my $stmd=int(&score_single($tmdif, $fulls_tmd, @tmdif)+0.5);
# score for len diff
my $lendif=abs($len2-$len1);
#print "lendif:\t", join("\t", $lendif, $fulls_lend, @lendif),"\n";
my $slend=int(&score_single($lendif, $fulls_lend, @lendif)+0.5);
## product
my $sprod=$fulls_prod;
my $dis;
if(!defined $NoSpecificity){
my ($pnum, $apeff, $apinfos);
($pnum, $apeff, $apinfos)=&get_highest_bound($product{$tid}{$tid}, 1000000, "Eff");
my ($peffs, $pinfos);
if($pnum!~/\+/ && $pnum<=3){
$peffs = join(",", @{$apeff});
$pinfos = join(";", @{$apinfos});
}else{
$peffs = join(",", @{$apeff}[0..2]);
$pinfos = join(";", @{$apinfos}[0..2]);
}
@{$prod_info{$tid}}=($pnum, $peffs, $pinfos);
# print "prod:\t", join("\t", $pnum, $peffs, $pinfos),"\n";
$sprod = &bound_score($pnum, $peffs, $fulls_prod, "Eff");
($dis) = $apinfos->[0]=~/^(\S+)\//;
if(defined $OutAllProduct){
print P ">$tid\t$pnum\n";
my @peff=@{$apeff};
my @pinfo=@{$apinfos};
for(my $i=0; $i<@{$apeff}; $i++){
print P join("\t", $apinfos->[$i], $apeff->[$i]),"\n";
}
}
}
# score for dis
#print "dis:\t", join("\t", $dis, $fulls_dis, @rdis),"\n";
my $sdis=int(&score_single($dis, $fulls_dis, @rdis)+0.5);
my $srel=$spos+$slend+$stmd+$sdis+$sprod;
my $stotal = $s1*$w1 + $s2*$w2 + $srel*$wr + $sp*$wp;
$stotal = sprintf("%.1f", $stotal);
@{$score_info{$tid}}=($stotal, join(",", $spos, $sdis, $slend, $stmd, $sprod));
}
foreach my $id(@ids){
my ($tid,$tp)=$id=~/(\S+)-([12FRP])$/;
$tp=~s/F/1/;
$tp=~s/R/2/;
my ($abase, $afeature, $ameth, $aspec, $bnumtm) = @{$info{$tid}{$tp}};
my @prods = @{$prod_info{$tid}};
if($tp eq "P"){
@prods=@{$prod_info{$id}};
}
print O join("\t", @{$abase}, @{$score_info{$tid}}, join("|",@{$score_info{$id}}), @{$afeature}, @{$ameth},@prods),"\n";
}
close(I);
close(O);
if(defined $OutAllProduct){
close(P);
}
}
## output cross product
if($etype eq "MultiPlex" && !defined $NoSpecificity){
open(C, ">$outdir/$fkey.final.cross.product") or die $!;
foreach my $tid1(sort {$a cmp $b} keys %product){
foreach my $tid2(sort {$a cmp $b} keys %{$product{$tid1}}){
my ($tid1_t)=$tid1=~/(\S+)-[UDP]-/;
my ($tid2_t)=$tid2=~/(\S+)-[UDP]-/;
$tid1_t = defined $tid1_t? $tid1_t: $tid1;
$tid2_t = defined $tid2_t? $tid2_t: $tid2;
next if($tid1_t eq $tid2_t);
my ($pnum, $apeff, $apinfos)=&get_highest_bound($product{$tid1}{$tid2}, 1000000, "Eff");
print C ">$tid1\_$tid2\t$pnum\n";
my @peff=@{$apeff};
my @pinfo=@{$apinfos};
for(my $i=0; $i<@{$apeff}; $i++){
print C join("\t", $apeff->[$i], $apinfos->[$i]),"\n";
}
}
}
close(C);
}
#######################################################################################
print STDOUT "\nDone. Total elapsed time : ",time()-$BEGIN_TIME,"s\n";
#######################################################################################
# ------------------------------------------------------------------
# sub function
# ------------------------------------------------------------------
sub USAGE {#
my $usage=<<"USAGE";
Program:
Version: $version
Contact:zeng huaping<huaping.zeng\@genetalks.com>
-io: oligo file can be two format as following, ID must like: xxx-F, xxx-R, xxx-P, xxx-1, xxx-2.
Common Format:
ID1-F AAAAAAAAAAAA
ID1-P AAAAAAAAAAAAAAAA
ID1-R AAAAAAAAAAAA
ID2-1 AAAAAAAAAAAA
ID2-P AAAAAAAAAAAAAAAA
ID2-2 AAAAAAAAAAAA
Evalue Format: xxx.final.result
#Chr Start Strand ID Seq Len Dis2Target ProductSize ScoreTotal ScorePair ScoreOligo Tm GC Hairpin DimerType DimerSize EndANum EndStability SNP Poly OligoBound BoundNum HighestTm HighestInfo
chr1 100379124 - rs113994132-B1-1 TTTTTCTGTTCCACTCATCATATGAGA 27 0 162 286 25,11,10,20,30 41|3,10,0,7,10,-5,5,0 58.90 0.333 55.89 7.91 20.73 1 -6.7 0D1:TTTTTCTGTTCCACTCATCATATGAGE 22T5 10|58.84,49.32,48.69 1 1.00 162/chr1,P1,-100379124,|||||||||||||||||||||||||||,58.84,P2,+100378962,|||||||||||||||||||||||||||||,59.58
chr1 100379097 - rs113994132-B1-P CCTTTATAGCCTTTCCTGAAAAATGACATAAGACATGGTA 40 40 0 286 30 63.5|1,3,10,5,10,10,3,7 66.39 0.350 30.67 -18.20 -6.10 2 0 NA 17A5,26T3,35T3 15|65.97,50.26,49.55 1 0.86 27/chr1,-100379097,||||||||||||||||||||||||||||||||||||||||,65.97:chr1,P1,-100379124,|||||||||||||||||||||||||||,58.84,P2,+100378962,|||||||||||||||||||||||||||||,59.58
-rd: distance range of pair primers, (best_min, best_max, min, max) separted by ",", example:
face-to-face: |---> P1 x dis_range: 120,160,80,200
(SNP) x P2 <---|
face-to-face: P1 |---> x dis_range:120,160,80,200
(Region) x <---| P2
________________
back-to-back: x <---| P2 dis_range:5,10,0,15
P1 |---> x (Overlap between p1 and p2: dis > 0)
__________
back-to-back: x <---| P2 dis_range:-50,-40,-60,-30
P1 |---> x (No overlap between p1 and p2: dis < 0)
_______________
Nested: P2 --->| x dis_range(P2-P1):-15,-10,-30,-3
P1 --->| x (dis < 0)
Usage:
Options:
-io <file> Input oligo file, Common or Evalue format(as shown in the above), forced
when -ib is given, must be Evalue format.
when -ib not given, must be Common format.
-ib <file> Input oligo's bounds file, evalue oligos when not given, optional
-id <files> Input database files separated by "," to evalue specificity, [$fdatabases]
-k <str> Key of output file, forced
-tp <str> primer type, "face-to-face", "back-to-back", "Nested", ["face-to-face"]
-ep <str> evalue type, "SinglePlex", "MultiPlex", ["SinglePlex"]
-mp <int> maximum products number to be caculated, to reduce running time. [$max_prodn]
--Methylation Design methylation oligos
--NoSpecificity Not evalue specificity
--AllEvalue Evalue all possible products, not only F<->R, contain F<->F and R<->R.
--OutAllProduct Output All Product Info.
-td <int> thread in bwa, [$thread]
-od <dir> Dir of output file, default ./
Caculating efficiency options:
-sz <int> max PCR fragment size, [$PCRsize]
-tm <int> optimal tm of primer, [$opt_tm]
-tmb <int> optimal tm of probe, [$opt_tm_probe]
-rd <str> distance range of pair primers, (best_min, best_max, min, max) separted by ",", [$range]
-stm <int> min tm to amplify when caculate specifity, [$min_tm_spec]
-me <float> min efficiency to consider a product, [$min_eff]
-h Help
USAGE
print $usage;
exit;
}