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1st_step+Adapter.pl
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1st_step+Adapter.pl
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#!uer/bin/perl
use warnings;
use strict;
use File::Basename qw(basename dirname);
use FindBin qw($Bin $Script);
use Getopt::Long;
sub usage{
print <<USAGE;
usage:
perl $0 -listMode1 listfile -report -project -prefix -QCRaw -Resequencelist -outdir -QN -exists
1. -listMode1 listfile
Column 1:sampleName
Column 2:sample library
Column 3:the time of beginning sequence
Column 4:Flowcell number
Column 5:lane
Column 6:Rawdir(eg:/share/fqdata032/solexa)
2. -report the path of report output
3. -prefix 1
4. -project projectname(eg:HK11165_HUMqvwR)
5. -help
6. -QCRaw
7. -Resequencelist
8. -NRate 3-10
9. -maxErrorRate 2
10.-contGC 37-49
11. -maxproGC 0.1
12. -minQ20 83
13. -minQ30 75
14. -librarylist
15. -outdir
16. -Nnumber 5
17. -Lowquality 0.5
18. -QN
19. -listMode2 listfile
Column 1:lanelib
Column 2:Rawdir
20. -finsih
USAGE
}
#===========================parameters===========================#
my($help,$listMode1,$listMode2,$prefix,$project,$report,$QCRaw,$Resequencelist,$NRate,$maxErrorRate,$contGC,$maxproGC,$minQ20,$minQ30,$librarylist,$QN,$finish);
my($FilterRawdir,$Nnumber,$Lowquality,$outdir);
my%hashexists;
GetOptions(
"help"=>\$help,
"listMode1:s"=>\$listMode1,
"listMode2:s"=>\$listMode2,
"librarylist:s"=>\$librarylist,
"report:s"=>\$report,
"project:s"=>\$project,
"prefix:s"=>\$prefix,
"QCRaw"=>\$QCRaw,
"Resequencelist"=>\$Resequencelist,
"NRate:s"=>\$NRate,
"maxErrorRate:s"=>\$maxErrorRate,
"contGC:s"=>\$contGC,
"maxproGC:s"=>\$maxproGC,
"minQ20:s"=>\$minQ20,
"minQ30:s"=>\$minQ30,
"FilterRawdir:s"=>\$FilterRawdir,
"outdir:s"=>\$outdir,
"Nnumber:s"=>\$Nnumber,
"Lowquality:s"=>\$Lowquality,
"QN"=>\$QN,
"finish:s"=>\$finish,
);
if($help){ &usage();exit 0;}
unless(defined $project){&usage();exit 0;}
unless(defined $prefix){$prefix="01"};
if($QCRaw){
unless (defined $report){$report="./"};
# unless (defined $FilterRawdir){$FilterRawdir="./FilterRawdir";mkdir($FilterRawdir)||die $!};
}
if($Resequencelist){
unless (defined $outdir){$outdir="./"};
unless (defined $Nnumber){$Nnumber="5"};
unless (defined $Lowquality ){$Lowquality="0.5"};
}
#===============================================default==============================================#
if($Resequencelist){
my$Resequencelistfile="$outdir/$prefix.$project.RE.list";
open RESE,">","$Resequencelistfile" or die $!;
}
if($finish){
open EX,"$finish" or die $!;
while(<EX>){
chomp;
my$existslane=(split /\s+/,$_)[0];
$hashexists{$existslane}=1;
}
}
#===========================================do QC Rawdata============================================#
my($QCRawreport,$mincontGC,$maxcontGC,$firstNRate,$secondNRate);
if($QCRaw){
my$QCRawreport="$report/$prefix.$project.QC_raw.report";
my$disqualification="$report/$prefix.$project.disqualification";
open DIS,">","$disqualification" or die $!;
open QCRAWRE,">","$QCRawreport" or die $!;
unless (defined $NRate){$NRate="3-10"};
unless (defined $contGC){$contGC="37-49"};
unless (defined $maxErrorRate){$maxErrorRate="2"};
unless (defined $maxproGC){$maxproGC="0.1"};
unless (defined $minQ20){$minQ20="83"};
unless (defined $minQ30){$minQ30="75"};
($mincontGC,$maxcontGC)=(split /\-/,$contGC)[0,1];
($firstNRate,$secondNRate)=(split /\-/,$NRate)[0,1];
print QCRAWRE"Samplename\tPath\tN_Rate($firstNRate%-$secondNRate%)\tError($maxErrorRate%)\tcontentGC($mincontGC%-$maxcontGC%)\tproportionGC($maxproGC)\tQ20($minQ20%)\tQ30($minQ30%)\tparticulars\n";
}
#==============================================Main process===========================================#
my($sampleName,$samplelibrary,$Flowcell,$lane,$Rawdir,$time,$RawPath);
my%hashlib;
if($listMode1){
open LIST,"$listMode1" or die $!;
}elsif($listMode2){
if($librarylist){
open LIB,"$librarylist" or die $!;
open LIST,"$listMode2" or die $!;
while(<LIB>){
chomp;
my($l_sample,$l_library)=(split /\t/,$_)[0,1];
unless(exists $hashlib{$l_library}){
$hashlib{$l_library}=$l_sample;
}
}
}else{
print "WARNING: Need the file of library information list ,you can get from BMS!\n";
&usage();exit 0;
}
}
#=========================================================================================
while(<LIST>){
chomp;
my@list=split;
if($listMode1){
$sampleName=$list[0];
$samplelibrary=$list[1];
$time=$list[2];
$Flowcell=$list[3];
$lane=$list[4];
$Rawdir=$list[5];
$RawPath="$Rawdir/$project/*/*\_$Flowcell\_L$lane\_$samplelibrary/*\_$Flowcell\_L$lane\_$samplelibrary\_1.fq.gz";
}elsif($listMode2){
my$lanelibT=$list[0];
($time,$Flowcell,$lane,$samplelibrary)=(split /\_/,$lanelibT)[0,2,3,4];
$lane=~s/L//g;
$Rawdir=$list[1];
if(exists $hashlib{$samplelibrary}){
$sampleName=$hashlib{$samplelibrary};
}else{
print "WARNING:the library is wrong\n";
}
$RawPath="$Rawdir/$project/*/$lanelibT/$lanelibT\_1.fq.gz";
}
my@fqlist = glob($RawPath);
if(@fqlist==0){
print "WARNING:No FASTQ file for $sampleName,$samplelibrary,$Flowcell,$Rawdir/$project\n";
next;
}
foreach my$path(@fqlist){
my$fq_gz=basename($path);my@temp=split(/\_/,$fq_gz);my$lanelib=join"_",@temp[0..4];
if($finish){
if(exists $hashexists{$lanelib}){
print "the $lanelib have been exists\n";
next;
}
}
my$rawLaneDir=dirname($path);
my$pooling=basename(dirname($rawLaneDir));
my$projectT=basename(dirname(dirname($rawLaneDir)));
my$raw_pe1="$rawLaneDir/$lanelib\_1.fq.gz";
my$raw_pe2="$rawLaneDir/$lanelib\_2.fq.gz";
my$adapterlist1="$rawLaneDir/1.adapter.list.gz";
my$adapterlist2="$rawLaneDir/2.adapter.list.gz";
if($Resequencelist){
unless($QCRaw){
print RESE"$sampleName\t$raw_pe1\t$raw_pe2\t$adapterlist1\t$adapterlist2\t$lanelib\t$Nnumber\t$Lowquality\n";}
}
#==================================================QC Rawfile==========================================#
if($QCRaw){
my($NRateflag,$error_rateflag,$contGCflag,$maxproGCflag,$minQ20flag,$minQ30flag)=(0,0,0,0,0,0);
my($NRatevalue,$errorvalue,$contGCvalue,$maxproGCvalue,$minQ20value,$minQ30value)=(0,0,0,0,0,0);
my($GC_number,$position_number)=(0,0);
my@Nlist1=();
my@Nlist2=();
my@Ntemp=();
my$Nflag;
my$check_pe1="$rawLaneDir/1.fqcheck";
my$check_pe2="$rawLaneDir/2.fqcheck";
my@particulars=("congratulations");
open CH1,"$check_pe1" or die $!;
open CH2,"$check_pe2" or die $!;
while(<CH1>){
chomp;
if($_=~/^base/){
$position_number++;
my@check1=split;
my$pos1=$check1[1]-1;
my$GC_D_value1=abs($check1[3]-$check1[4]);
my$AT_D_value1=abs($check1[2]-$check1[5]);
if($GC_D_value1>2 or $AT_D_value1>2){
$GC_number++;
}
if($check1[6]>=$firstNRate and $check1[6]<$secondNRate){
push @Nlist1,$pos1;
if($NRateflag==0){
$NRatevalue=$check1[6];
$NRateflag=1;
}else{
my($maxNRatevalue)=sort{$b <=> $a}($NRatevalue,$check1[6]);
$NRatevalue=$maxNRatevalue;
}
}elsif($check1[6]>$secondNRate){
push @Nlist1,$pos1;
$NRateflag=2;
my($maxNRatevalue)=sort{$b <=> $a}($NRatevalue,$check1[6]);
$NRatevalue=$maxNRatevalue;
}
}elsif($_=~/^Error/){
my$QC=<CH1>;
my($error_rate,$GC,$Q20,$Q30)=(split /\s+/,$QC)[0,1,2,3];
if($error_rate>$maxErrorRate){
$errorvalue=$error_rate;
$error_rateflag=1;
}
if($GC>$maxcontGC or $GC<$mincontGC ){
$contGCvalue=$GC;
$contGCflag=1;
}
if($Q20<$minQ20 ){
$minQ20value=$Q20;
$minQ20flag=1;
}
if($Q30<$minQ30 ){
$minQ30value=$Q30;
$minQ30flag=1;
}
}
}
while(<CH2>){
chomp;
if($_=~/^base/){
$position_number++;
my@check2=split;
my$pos2=$check2[1]-1;
my$GC_D_value2=abs($check2[3]-$check2[4]);
my$AT_D_value2=abs($check2[2]-$check2[5]);
if($GC_D_value2>2 or $AT_D_value2>2){
$GC_number++;
}
if($check2[6]>=$firstNRate and $check2[6]<$secondNRate){
push @Nlist2,$pos2;
if($NRateflag==0){
$NRatevalue=$check2[6];
$NRateflag=1;
}else{
my($maxNRatevalue)=sort{$b <=>$a}($NRatevalue,$check2[6]);
$NRatevalue=$maxNRatevalue;
}
}elsif($check2[6]>$secondNRate){
push @Nlist2,$pos2;
$NRateflag=2;
my($maxNRatevalue)=sort{$b <=>$a}($NRatevalue,$check2[6]);
$NRatevalue=$maxNRatevalue;
}
}elsif($_=~/^Error/){
my$QC=<CH2>;
my($error_rate,$GC,$Q20,$Q30)=(split /\s+/,$QC)[0,1,2,3];
if($error_rate>$maxErrorRate){
my($maxerrorvalue)=sort{$b <=> $a}($error_rate,$errorvalue);
$errorvalue=$maxerrorvalue;
$error_rateflag=1;
}
if($GC>$maxcontGC or $GC<$mincontGC){
if($contGCflag==1){
my$temp=$contGCvalue;
$contGCvalue=join":",$temp,$GC;
}
}
if($Q20<$minQ20){
if($minQ20flag==1){
my($minQ20)=sort{$a<=>$b}($Q20,$minQ20value);
$minQ20value=$minQ20;
}else{
$minQ20value=$Q20;
$minQ20flag=1;
}
}
if($Q30<$minQ30){
if($minQ30flag){
my($minQ30)=sort{$a<=>$b}{$Q30,$minQ30value};
$minQ30value=$minQ30;
}else{
$minQ30value=$Q30;
$minQ30flag=1;
}
}
}
}
my$Nlisttemp1=join ",",@Nlist1;my$Nlisttemp2=join ",",@Nlist2;
if(@Nlist1){my$Nlist_fq1=join":",1,$Nlisttemp1;push @Ntemp,$Nlist_fq1;}
if(@Nlist2){my$Nlist_fq2=join":",2,$Nlisttemp2;push @Ntemp,$Nlist_fq2;}
if(@Ntemp){
$Nflag=join ";",@Ntemp;
}
my$proGC=$GC_number/$position_number;
if($proGC>$maxproGC){
$maxproGCvalue=$proGC;
$maxproGCflag=1;
}
if($NRateflag or $error_rateflag or $contGCflag or $minQ20flag or $minQ30flag or $maxproGCflag){
if($NRateflag){
print DIS"$sampleName\t$lanelib\t$Rawdir\t$Nflag\n";
}else{
print DIS"$sampleName\t$lanelib\t$Rawdir\ttemp\n";
if($QN){
print RESE"$sampleName\t$raw_pe1\t$raw_pe2\t$adapterlist1\t$adapterlist2\t$lanelib\t$Nnumber\t$Lowquality\n";
}
}
my@particulars;
if($NRateflag==1){
push @particulars,"you just need remove all of the N base that in the fastq";
}elsif($NRateflag==2){
push @particulars,"you need the tile !!";
}else{
#push @particulars,"the rate of N base is ok .";
$NRatevalue="OK";
}
if($error_rateflag){
push @particulars,"Unfortunately,the rate of error base is too high";
}else{
#push @particulars,"the Error rate is too ok.";
$errorvalue="OK";
}
if($maxproGCflag){
push @particulars,"the GC is unequal";
}else{
$maxproGCvalue="OK";
}
if($contGCflag){
push @particulars,"the content of GC is unreasonable.Maybe the data is unavailable";
}else{
#push @particulars,"the content of GC is reasonable .";
$contGCvalue="OK";
}
if($minQ20flag){
push @particulars,"Q20 is disqualification";
}else{
#push @particulars,"Q20 is ok .";
$minQ20value="OK";
}
if($minQ30flag){
push @particulars,"Q30 is disqualification";
}else{
#push @particulars,"Q30 is ok .";
$minQ30value="OK";
}
if($maxproGCflag){
push @particulars,"GC is unequal";
}else{
#push @particulars,"the proportion of GC is ok .";
$maxproGCvalue="OK";
}
my$particulars_list=join ";",@particulars;
print QCRAWRE"$sampleName\t$lanelib\t$NRatevalue\t$errorvalue\t$contGCvalue\t$maxproGCvalue\t$minQ20value\t$minQ30value\tUnfortunately\n";
print "$particulars_list\n";
}else{
print QCRAWRE"$sampleName\t$lanelib\tOK\tOK\tOK\tOK\tOK\tOK\tcongratulations\n";
if($Resequencelist ){
print RESE"$sampleName\t$raw_pe1\t$raw_pe2\t$adapterlist1\t$adapterlist2\t$lanelib\t$Nnumber\t$Lowquality\n";
}
}
}
#==========================================================================================================#
}
}