perl怎么对组装结果中GAP左右批量设计引物

#!/usr/bin/perl -w use strict; use Cwd qw(abs_path getcwd); use Getopt::Long; use Data::Dumper; use FindBin qw($Bin $Script); use File::Basename qw(basename dirname); use Bio::SeqIO; my $BEGIN_TIME=time(); my $version="1.0"; # ------------------------------------------------------------------ # GetOptions # ------------------------------------------------------------------ my ($fa,$flank,$epcrfa,$od,$cut,$outprefix,$PRIMER_NUM_RETURN,$PRIMER_OPT_SIZE,$PRIMER_MIN_SIZE,$PRIMER_MAX_SIZE,$PRIMER_PRODUCT_SIZE_RANGE,$PRIMER_MAX_NS_ACCEPTED); GetOptions( "help|?" =>\&USAGE, "fa=s"=>\$fa, "flank=s"=>\$flank, "PRIMER_MIN_SIZE=s"=>\$PRIMER_MIN_SIZE, "PRIMER_OPT_SIZE=s"=>\$PRIMER_OPT_SIZE, "PRIMER_MAX_SIZE=s"=>\$PRIMER_MAX_SIZE, "PRIMER_NUM_RETURN=s"=>\$PRIMER_NUM_RETURN, "PRIMER_PRODUCT_SIZE_RANGE=s"=>\$PRIMER_PRODUCT_SIZE_RANGE, "epcrfa=s"=>\$epcrfa, "od=s"=>\$od, "prefix=s"=>\$outprefix, ) or &USAGE; &USAGE unless($fa); sub USAGE { my $usage=<<"USAGE"; Program: $0 Version: $version Contact: huang2002003\@126.com Discription: Usage: Options: -fa Input fa file, forced -flank 100 cut 100 -PRIMER_MIN_SIZE 18 Minimum acceptable length of a primer. Must be greater than 0 and less than or equal to PRIMER_MAX_SIZE -PRIMER_OPT_SIZE 20 Optimum length (in bases) of a primer. Primer3 will attempt to pick primers close to this length. -PRIMER_MAX_SIZE 23 Maximum acceptable length (in bases) of a primer. Currently this parameter cannot be larger than 35. This limit is governed by maximum oligo size for which primer3's melting-temperature is valid. -PRIMER_PRODUCT_SIZE_RANGE 100-300 The associated values specify the lengths of the product that the user wants the primers to create, and is a space separated list of elements of the form -PRIMER_NUM_RETURN 1 Total num primer to search -epcrfa run E-PCR to filter multi mapped primers; required; -od Key of output dir,default cwd; -prefix defoult demo; -h Help USAGE print $usage; exit 1; } $PRIMER_MIN_SIZE||=18; $PRIMER_OPT_SIZE||=20; $PRIMER_MAX_SIZE||=23; $PRIMER_NUM_RETURN||=1; $PRIMER_MAX_NS_ACCEPTED||=1; $PRIMER_PRODUCT_SIZE_RANGE||="100-300"; $od||=getcwd; $flank||=100; $od=abs_path($od); unless(-d $od){ mkdir $od;} $outprefix||="SSR"; $fa=abs_path($fa); $epcrfa||=$fa; $epcrfa=abs_path($epcrfa); ################################################################# my%ref=(); my%ref_len=(); my$in = Bio::SeqIO->new(-file => "$fa" , -format => 'Fasta'); while ( my $seq = $in->next_seq() ) { $ref{$seq->id}=$seq; $ref_len{$seq->id}=$seq->length; } $in->close(); ############find SSR by misa####################### print "perl /biosoft/MISA/my_misa1.pl $fa $od/$outprefix.misa $od/$outprefix.misa.statistics\n"; system("perl /biosoft/MISA/my_misa1.pl $fa $od/$outprefix.misa $od/$outprefix.misa.statistics"); ###########################primer design ############################### open IN,"$od/$outprefix.misa" or die "$!"; open OUT,">$od/primer3.input" or die "$!"; my%ssr_pos=(); #ssr相关信息 my%SSR=(); # SSR type while(my$line=){ chomp $line; my@tmp=split(/\t/,$line); next if ($tmp[0] eq "ID"); my$ID="$tmp[0]_$tmp[5]_$tmp[6]_$tmp[4]"; $ssr_pos{$ID}="$tmp[0]\t$tmp[5]\t$tmp[6]\t$tmp[4]\t$tmp[2]"; my$seq=&get_target_fa($tmp[0],$tmp[5]-$flank,$tmp[6]+$flank); #print $seq."\n"; my$tar=0; if(($tmp[5]-$flank)<=0){ $tar=$tmp[5]; }else{ $tar=$flank; } if ($seq){ $SSR{$ID}=$tmp[3]; print OUT "SEQUENCE_ID=$ID\n"; print OUT "SEQUENCE_TEMPLATE=$seq\n"; printf OUT ("SEQUENCE_TARGET=%d,%d\n",$tar+1,$tmp[4]); print OUT "SPRIMER_TASK=pick_detection_primers\n"; print OUT "PRIMER_PICK_LEFT_PRIMER=1\n"; print OUT "PRIMER_PICK_RIGHT_PRIMER=1\n"; print OUT "PRIMER_NUM_RETURN=$PRIMER_NUM_RETURN\n"; print OUT "PRIMER_MAX_END_STABILITY=250\n"; print OUT "PRIMER_MIN_SIZE=$PRIMER_MIN_SIZE\n"; print OUT "PRIMER_OPT_SIZE=$PRIMER_OPT_SIZE\n"; print OUT "PRIMER_MAX_SIZE=$PRIMER_MAX_SIZE\n"; print OUT "PRIMER_MAX_NS_ACCEPTED=1\n"; print OUT "PRIMER_MIN_GC=40.0\nPRIMER_MAX_GC=60.0\n"; print OUT "PRIMER_PRODUCT_SIZE_RANGE=$PRIMER_PRODUCT_SIZE_RANGE\n"; printf OUT ("SEQUENCE_INTERNAL_EXCLUDED_REGION=%d,%d\n",$tar+1,$tmp[4]); print OUT "=\n"; } } close(OUT); close(IN); print "/biosoft/Primer3/primer3-2.3.7/src/primer3_core -p3_settings_file=/biosoft/Primer3/primer3-2.3.7/default_settings.txt -output=$od/$outprefix.primers $od/primer3.input\n"; system("/biosoft/Primer3/primer3-2.3.7/src/primer3_core -p3_settings_file=/biosoft/Primer3/primer3-2.3.7/default_settings.txt -output=$od/$outprefix.primers $od/primer3.input"); ###############################e-PCR ##################### my%Pseq=(); $/="=\n"; open IN ,"$od/$outprefix.primers" or die "$!"; open OUT,">$od/$outprefix.primers.xls" or die "$!"; print OUT "#SEQUENCE_ID\tPRIMER_LEFT_SEQUENCE\tPRIMER_RIGHT_SEQUENCE\tPRIMER_PAIR_PRODUCT_SIZE\tPRIMER_LEFT_TM\tPRIMER_RIGHT_TM\tPRIMER_LEFT_GC_PERCENT\tPRIMER_RIGHT_GC_PERCENT\tSSR\tSSR_TYPE\n"; while(my$line=){ chomp$line; my@field=split(/\n/,$line); my%primers=&get_hash(@field); next if ! defined($primers{"PRIMER_PAIR_NUM_RETURNED"}) or $primers{"PRIMER_PAIR_NUM_RETURNED"}==0; if($primers{"PRIMER_PAIR_NUM_RETURNED"}>=1){ for my$i (0..($primers{"PRIMER_PAIR_NUM_RETURNED"}-1)){ $Pseq{"$primers{SEQUENCE_ID}"}{$i}=[$primers{"PRIMER_LEFT_${i}_SEQUENCE"},$primers{"PRIMER_RIGHT_${i}_SEQUENCE"}]; print OUT join("\t",("$primers{SEQUENCE_ID}_${i}",$primers{"PRIMER_LEFT_${i}_SEQUENCE"},$primers{"PRIMER_RIGHT_${i}_SEQUENCE"},$primers{"PRIMER_PAIR_${i}_PRODUCT_SIZE"},$primers{"PRIMER_LEFT_${i}_TM"},$primers{"PRIMER_RIGHT_${i}_TM"},$primers{"PRIMER_LEFT_${i}_GC_PERCENT"},$primers{"PRIMER_RIGHT_${i}_GC_PERCENT"},$SSR{$primers{"SEQUENCE_ID"}}))."\n"; } } } $/="\n"; close(OUT); print "/biosoft/e-PCR/Linux-x86_64/e-PCR -w9 -f 1 -m100 $od/$outprefix.primers.xls D=100-400 $epcrfa N=1 G=1 T=3 > $od/$outprefix.epcr.out\n"; system("/biosoft/e-PCR/Linux-x86_64/e-PCR -w9 -f 1 -m100 $od/$outprefix.primers.xls D=100-400 $epcrfa N=1 G=1 T=3 > $od/$outprefix.epcr.out"); ###################################################################################### open IN ,"$od/$outprefix.epcr.out" or die "$!"; open OUT,">$od/$outprefix.primers.result.xls" or die "$!"; my %P=(); while(my$line=){ chomp$line; my@tmp=split(/\t/,$line); next if $tmp[6]+$tmp[7] >1; $tmp[5]=~s#/.*$##; my$ssr_id=$tmp[1]; my$num=0; ($ssr_id,$num)=($ssr_id=~/(.*)_(\d+)$/); $P{$tmp[1]}{"$tmp[1]_$tmp[3]_$tmp[4]"}=[$tmp[1],$ssr_pos{$ssr_id},$Pseq{$ssr_id}{$num}->[0],$Pseq{$ssr_id}{$num}->[1],$tmp[5],@tmp[6..$#tmp]]; } close(IN); print OUT "#GAP_ID\tCHR\tGAP_START\tGAP_END\tGAP_LENGTH\tGAP_TYPE\tPRIMER_LEFT_SEQUENCE\tPRIMER_RIGHT_SEQUENCE\tPRIMER_PAIR_PRODUCT_SIZE/PREDICT_RANGE\tMISM\tGAPS\tPRIMER_LEFT_TM\tPRIMER_RIGHT_TM\tPRIMER_LEFT_GC_PERCENT\tPRIMER_RIGHT_GC_PERCENT\tGAP\n"; open OUT1,">$od/$outprefix.NOprimers.result.xls" or die "$!"; for my $id (sort keys %SSR){ for my$i(0..($PRIMER_NUM_RETURN-1)){ if (exists $P{"${id}_$i"}){ my@ps=(keys %{$P{"${id}_$i"}}); if(@ps ==1){ print OUT join("\t",@{$P{"${id}_$i"}{$ps[0]}})."\n"; } }else{ print OUT1 "${id} not primers\n"; } } } close(OUT); close(OUT1); open OUT,">$od/readme.txt" or die "$!"; my $readme=<<"README"; 建议使用editplus打开本文件； *primers.result.xls #SEQUENCE_ID-- 引物ID （命名规则：GAP所在来源序列_GAP所在位置start_GAP所在位置end_GAP长度_备用引物编号 ; ） PRIMER_LEFT_SEQUENCE-- 左引物 PRIMER_RIGHT_SEQUENCE-- 右引物 PRIMER_PAIR_PRODUCT_SIZE/PREDICT_RANGE-- MISM-- Total number of mismatches for two primers(ePCR) GAPS-- Total number of gaps for two primers(ePCR) PRIMER_LEFT_TM-- 退火温度 PRIMER_RIGHT_TM-- 退火温度 PRIMER_LEFT_GC_PERCENT-- GC含量 PRIMER_RIGHT_GC_PERCENT-- GC含量 GAP-- GAP类型方法： 2.提取GAP左右及其左右序列，用primer3设计引物[2] 3.用Epcr ，在fasta上电子PCR，去除有多处比较的引物,保证引物扩增的特异性[3] [1] MISA:Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.). [2] Primer3: Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG (2012) Primer3 - new capabilities and interfaces. Nucleic Acids Research 40(15):e115 [3] http://www.ncbi.nlm.nih.gov/tools/epcr/ README print OUT $readme; close(OUT); ################################################################################ sub get_hash{ my@info=@_; my%result=(); for my$i(@info){ next if $i=~/^\s*$/; my($k,$v)=split(/=/,$i); $result{$k}=$v; } return %result; } sub get_target_fa(){ my $id=shift; my $posU=shift; my $posD=shift; if($posU<=0){ $posU=1; } my $seqobj=$ref{$id}; my $len=$ref_len{$id}; return $seqobj->subseq($posU,$len) if $posD>$len; my $tri=""; $tri=$seqobj->subseq($posU,$posD); return $tri; }

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