/
fqc.pl
1191 lines (1025 loc) · 34.8 KB
/
fqc.pl
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#!/usr/bin/perl -w
use strict;
my $version=1.05;
use Getopt::Long;
my %opts;
if($ARGV[0] eq "-v" or $ARGV[0] eq "--help"){
print STDERR "$0, Version $version\n";
exit;
}
&command if(@ARGV<1);
GetOptions(\%opts,"i=s","f=s","r=s","o=s","a=s","q:s","c:s","l:s","p","v","h");
if (!(defined $opts{i} || defined $opts{f} and defined $opts{r}) and !defined $opts{o} || defined $opts{h}) {
&PrintUsage;
}
my $file1 = $opts{'i'}; #Single Reads file
my $file2 = $opts{'f'}; #forward fastq file
my $file3 = $opts{'r'}; #reverse fastq file
my $file4 = $opts{'o'}; #Output prefix
my $qCutOff = (defined $opts{c})?$opts{c}:20; #quality_threshold
my $format = (defined $opts{q})?$opts{q}:33; #Default:33
my $lenCut = (defined $opts{l})?$opts{l}:30; #Default:30
my $adapter = (defined $opts{a})?$opts{a}:"ATCGGAAGAGC"; #Default:Tru-Seq Adapter
my ($size1,$size2,$trimStart1,$trimStart2,$id1,$seq1,$qual1,$id2,$seq2,$qual2,@qual1,@qual2,$total_length1,$total_length2,$single_length1,$single_length2,$total_length_trimmed1,$total_length_trimmed2,$trimCount1,$trimCount2,$len1,$len2,$length1,$length2,$input,$high1,$high2,$high3,$high4,%hash,$pos,$pos1,$pos2,$adp,$dup_num);
$total_length1 = $total_length2 = $total_length_trimmed1 = $total_length_trimmed2 = $single_length1 = $single_length2 = 0;
my $oldtime = time();
my $cmd =$ARGV[0];
my %cmd_hash =(adapter=>\&adapter,quality=>\&quality,adp_dup=>\&adp_dup,adp_qual=>\&adp_qual,duplication=>\&duplication,all=>\&all);
if(($ARGV[0] eq "adapter") or ($ARGV[0] eq "quality") or ($ARGV[0] eq "adp_dup") or ($ARGV[0] eq "adp_qual") or ($ARGV[0] eq "all") or ($ARGV[0] eq "duplication")){
&{$cmd_hash{$cmd}};
}else{
&command;
}
#########################adapter#########################################
sub adapter {
open STAT,">$file4.stat.xls";
if(defined $opts{p}){
print STAT "Progrom,Parameters,Input and Output information:\n";
print STAT "$0 adapter -f $file2 -r $file3 -a $adapter -l $lenCut -p -o $file4\n";
print STAT "Input file:$file2 and $file3\n";
print STAT "Adapter sequence: $adapter\n";
print STAT "Output file: $file4"."_1.fastq and $file4"."_2.fastq\n";
print STAT "Output stat file: $file4"."_report.xls\n\n";
}else{
print STAT "$0 adapter -i $file1 -c $qCutOff -q $format -a $adapter -l $lenCut -o $file4\n";
print STAT "Input file:$file1\n";
print STAT "Adapter sequence: $adapter\n";
print STAT "Output file: $file4.fastq\n";
print STAT "Output stat file: $file4"."_report.xls\n\n";
}
if(defined $opts{p}){
open IN1,"$file2" || die $!;
open IN2,"$file3" || die $!;
open OUT1,">$file4"."_1.fastq" || die $!;
open OUT2,">$file4"."_2.fastq" || die $!;
while(!eof(IN1) && !eof(IN2)){
$id1 = <IN1>; $seq1 = <IN1>; <IN1>; $qual1 = <IN1>; $input++;
$id2 = <IN2>; $seq2 = <IN2>; <IN2>; $qual2 = <IN2>;
chomp ($id1,$seq1,$qual1,$id2,$seq2,$qual2);
if($seq1 =~ m/$adapter/ && $seq2 =~ m/$adapter/){
$pos1 = index($seq1,$adapter);
$pos2 = index($seq2,$adapter);
if($pos1 < $lenCut or $pos2 < $lenCut){
$adp++;
next;
}else{
$seq1 = substr($seq1,0,$pos1 - 2);
$qual1 = substr($qual1,0,$pos1 - 2);
$seq2 = substr($seq2,0,$pos2 - 2);
$qual2 = substr($qual2,0,$pos2 - 2);
print OUT1 "$id1\n$seq1\n+\n$qual1\n";
print OUT2 "$id2\n$seq2\n+\n$qual2\n";
}
}elsif($seq1 =~ m/$adapter/){
$pos1 = index($seq1,$adapter);
if($pos1 < $lenCut){
$adp++;
next;
}else{
$seq1 = substr($seq1,0,$pos1 - 2);
$qual1 = substr($qual1,0,$pos1 - 2);
print OUT1 "$id1\n$seq1\n+\n$qual1\n";
print OUT2 "$id2\n$seq2\n+\n$qual2\n";
}
}elsif($seq2 =~ m/$adapter/){
$pos2 = index($seq2,$adapter);
if($pos2 < $lenCut){
$adp++;
next;
}else{
$seq2 = substr($seq2,0,$pos2 - 2);
$qual2 = substr($qual2,0,$pos2 - 2);
print OUT1 "$id1\n$seq1\n+\n$qual1\n";
print OUT2 "$id2\n$seq2\n+\n$qual2\n";
}
}else{
print OUT1 "$id1\n$seq1\n+\n$qual1\n";
print OUT2 "$id2\n$seq2\n+\n$qual2\n";
}
}
close IN1;
close IN2;
close OUT1;
close OUT2;
}else{
open IN,"$file1" || die $!;
open OUT,">$file4"."_1.fastq" || die $!;
while(!eof(IN)){
$id1 = <IN>; $seq1 = <IN>; <IN>; $qual1 = <IN>; $input++;
chomp ($id1,$seq1,$qual1);
if($seq1 =~ m/$adapter/){
$pos = index($seq1,$adapter);
if($pos < $lenCut){
$adp++;
next;
}else{
$seq1 = substr($seq1,0,$pos - 2);
$qual1 = substr($qual1,0,$pos - 2);
print OUT "$id1\n$seq1\n+\n$qual1\n";
}
}else{
print OUT "$id1\n$seq1\n+\n$qual1\n";
}
}
close IN;
close OUT;
}
my $oo = time() - $oldtime;
my $reads = $input - $adp;
my $percentage = int($reads/$input*10000)/100;
if(defined $opts{p}){
print STAT "Name\tTotal_Reads\tValid_Data\tPercentage\n";
print STAT "$file2\t$input\t$reads\t$percentage%\n";
print STAT "$file2\t$input\t$reads\t$percentage%\n";
print STAT "\nTime consumption is $oo";
}else{
print STAT "Name\tTotal_Reads\tValid_Data\tPercentage\n";
print STAT "$file1\t$input\t$reads\t$percentage%\n";
print STAT "\nTime consumption is $oo sec.";
}
close STAT;
}
#########################quality#########################################
sub quality {
open STAT,">$file4.stat.xls";
if(defined $opts{p}){
print STAT "Progrom,Parameters,Input and Output information:\n";
print STAT "$0 quality -f $file2 -r $file3 -c $qCutOff -q $format -l $lenCut -p -o $file4\n";
print STAT "Input file:$file2 and $file3\n";
print STAT "Low quality threshold:$qCutOff\n";
print STAT "Input file format:$format\n";
print STAT "Output file: $file4.$qCutOff"."_1.fastq and $file4.$qCutOff"."_2.fastq\n";
print STAT "Output stat file: $file4.stat.xls\n\n";
}else{
print STAT "$0 quality -i $file1 -c $qCutOff -q $format -l $lenCut -o $file4\n";
print STAT "Input file:$file1\n";
print STAT "Low quality threshold:$qCutOff\n";
print STAT "Input file format:$format\n";;
print STAT "Output file: $file4.$qCutOff.fastq\n";
print STAT "Output stat file: $file4.stat.xls\n\n";
}
if(defined $opts{p}){ #Paired-End Format
open IN1,"$file2" || die $!;
open IN2,"$file3" || die $!;
open OUT1,">$file4.$qCutOff"."_1.fastq" || die $!;
open OUT2,">$file4.$qCutOff"."_2.fastq" || die $!;
open OUT3,">$file4"."_single_1.fastq" || die $!;
open OUT4,">$file4"."_single_2.fastq" || die $!;
while(!eof(IN1) && !eof(IN2)){
$id1 = <IN1>; $seq1 = <IN1>; <IN1>; $qual1 = <IN1>; $input++;
$id2 = <IN2>; $seq2 = <IN2>; <IN2>; $qual2 = <IN2>;
chomp ($id1,$seq1,$qual1,$id2,$seq2,$qual2);
$length1 = length $seq1;
$length2 = length $seq2;
$total_length1 = $total_length1 + $length1;
$total_length2 = $total_length2 + $length2;
##Remove lower bases:
@qual1 = split (//, $qual1); @qual2 = split (//, $qual2);
$trimCount1 = $trimCount2 = $trimStart1 = $trimStart2 = 0;
for(my $i = $#qual1; $i > 0; $i--) {
my $val1 = ord($qual1[$i]) - $format;
if($val1 < $qCutOff) {
$trimCount1++;
}else{
last;
}
}
for(my $i = 0;$i < $#qual1; $i++) {
my $val1 = ord($qual1[$i]) - $format;
if($val1 < $qCutOff) {
$trimStart1++;
}else{
last;
}
}
for(my $i=$#qual2; $i>0; $i--) {
my $val2 = ord($qual2[$i]) - $format;
if($val2 < $qCutOff) {
$trimCount2++;
}else{
last;
}
}
for(my $i = 0;$i < $#qual2; $i++) {
my $val2 = ord($qual2[$i]) - $format;
if($val2 < $qCutOff) {
$trimStart2++;
}else{
last;
}
}
$size1 = $length1 - $trimStart1 - $trimCount1;
$qual1 = substr($qual1,$trimStart1,$size1);
$seq1 = substr($seq1,$trimStart1,$size1);
$size2 = $length2 - $trimStart2 - $trimCount2;
$qual2 = substr($qual2,$trimStart2,$size2);
$seq2 = substr($seq2,$trimStart2,$size2);
$len1 = length $seq1;
$len2 = length $seq2;
if(($len1 >= $lenCut) && ($len2 >= $lenCut)){
print OUT1 "$id1\n$seq1\n+\n$qual1\n";
print OUT2 "$id2\n$seq2\n+\n$qual2\n";
$total_length_trimmed1 = $total_length_trimmed1 + $len1;
$total_length_trimmed2 = $total_length_trimmed2 + $len2;
$high1++; $high2++;
}elsif($len1 >= $lenCut){
print OUT3 "$id1\n$seq1\n+\n$qual1\n";
$high3++;$single_length1 = $single_length1 + $len1;
}elsif($len2 >= $lenCut){
print OUT4 "$id2\n$seq2\n+\n$qual2\n";
$high4++;$single_length2 = $single_length2 + $len2;
}
}
close IN1;
close IN2;
close OUT1;
close OUT2;
close OUT3;
close OUT4;
}else{ #Single Format
open IN1,"$file1" || die $!;
open OUT1,">$file4.$qCutOff.fastq" || die $!;
while(!eof(IN1)){
$id1 = <IN1>; $seq1 = <IN1>; <IN1>; $qual1 = <IN1>; $input++;
chomp ($id1,$seq1,$qual1);
$length1 = length $seq1;
$total_length1 = $total_length1 + $length1;
##Remove lower bases:
@qual1 = split (//, $qual1);
$trimCount1 = $trimStart1 = 0;
for(my $i=$#qual1; $i>0; $i--) {
my $val = ord($qual1[$i]) - $format;
if($val < $qCutOff) {
$trimCount1++;
}else{
last;
}
}
for(my $i = 0;$i < $#qual1; $i++) {
my $val = ord($qual1[$i]) - $format;
if($val < $qCutOff) {
$trimStart1++;
}else{
last;
}
}
$size1 = $length1 - $trimStart1 - $trimCount1;
$qual1 = substr($qual1,$trimStart1,$size1);
$seq1 = substr($seq1,$trimStart1,$size1);
$len1 = length $seq1;
if($len1 >= $lenCut){
print OUT1 "$id1\n$seq1\n+\n$qual1\n";
$total_length_trimmed1 = $total_length_trimmed1 + $len1;
$high1++;
}
}
close IN1;
close OUT1;
}
if(defined $opts{p}){
my $rata_trimmed1 = int(10000*$total_length_trimmed1/$total_length1)/100;
my $rata_trimmed2 = int(10000*$total_length_trimmed2/$total_length2)/100;
my $single1 = int(10000*$single_length1/$total_length1)/100;
my $single2 = int(10000*$single_length2/$total_length2)/100;
print STAT "Remove lower bases information:\n";
print STAT "Name\tTotal_Reads\tTotal_bases\tHigh_quility_reads\tHigh_quility_bases\tSingle_Reads\tSingle_bases\n";
print STAT "$file2\t$input\t$total_length1\t$high1\t$total_length_trimmed1($rata_trimmed1%)\t$high3\t$single_length1($single1%)\n";
print STAT "$file3\t$input\t$total_length2\t$high2\t$total_length_trimmed2($rata_trimmed2%)\t$high4\t$single_length2($single2%)\n\n";
}else{
my $rata_trimmed1 = int(10000*$total_length_trimmed1/$total_length1)/100;
print STAT "Remove lower bases information:\n";
print STAT "Total reads: $input\n";
print STAT "Total bases: $total_length1\n";
print STAT "High quility reads: $high1\n";
print STAT "Total bases after trimming: $total_length_trimmed1($rata_trimmed1%)\n\n";
}
my $oo = time() - $oldtime;
$oo = int(100*$oo/60)/100;
print STAT "All completed successfully!\n\nTime consumption is $oo min";
close STAT;
}
#########################duplication#########################################
sub duplication {
open STAT,">$file4.stat.xls";
if(defined $opts{p}){
print STAT "Progrom,Parameters,Input and Output information:\n";
print STAT "$0 duplication -f $file2 -r $file3 -l $lenCut -p -o $file4\n";
print STAT "Input file:$file2 and $file3\n";
print STAT "Output file: $file4.dup"."_1.fastq and $file4.dup"."_2.fastq\n";
print STAT "Output stat file: $file4.stat.xls\n\n";
}else{
print STAT "$0 duplication -i $file1 -l $lenCut -o $file4\n";
print STAT "Input file:$file1\n";
print STAT "Output file: $file4.dup.fastq\n";
print STAT "Output stat file: $file4.stat.xls\n\n";
}
if(defined $opts{p}){ #Paired-End Format
open IN1,"$file2" || die $!;
open IN2,"$file3" || die $!;
open OUT1,">$file4.dup"."_1.fastq" || die $!;
open OUT2,">$file4.dup"."_2.fastq" || die $!;
while(!eof(IN1) && !eof(IN2)){
$id1 = <IN1>; $seq1 = <IN1>; <IN1>; $qual1 = <IN1>; $input++;
$id2 = <IN2>; $seq2 = <IN2>; <IN2>; $qual2 = <IN2>;
chomp ($id1,$seq1,$qual1,$id2,$seq2,$qual2);
##Remove duplication:
my $dup = substr($seq1,0,$lenCut).substr($seq2,0,$lenCut);
$hash{$dup}++;
if($hash{$dup} == 1){
print OUT1 "$id1\n$seq1\n+\n$qual1\n";
print OUT2 "$id2\n$seq2\n+\n$qual2\n";
}else{
$dup_num++;
next;
}
}
close IN1;
close IN2;
close OUT1;
close OUT2;
}else{ #Single Format
open IN1,"$file1" || die $!;
open OUT1,">$file4.dup.fastq" || die $!;
while(!eof(IN1)){
$id1 = <IN1>; $seq1 = <IN1>; <IN1>; $qual1 = <IN1>; $input++;
chomp ($id1,$seq1,$qual1);
##Remove duplication:
my $dup = substr($seq1,0,$lenCut);
$hash{$dup}++;
if($hash{$dup} == 1){
print OUT1 "$id1\n$seq1\n+\n$qual1\n";
}else{
$dup_num++;
next;
}
}
close IN1;
close OUT1;
}
my $dup_rate = int(10000*$dup_num/$input)/100;
print STAT "Duplication information:\n";
print STAT "Total reads: $input\n";
print STAT "Duplicate reads: $dup_num\n";
print STAT "Duplication: $dup_rate%\n\n";
undef %hash;
my $oo = time() - $oldtime;
$oo = int(100*$oo/60)/100;
print STAT "All completed successfully!\n\nTime consumption is $oo min";
close STAT;
}
#########################adp_qual#########################################
sub adp_qual {
open STAT,">$file4.stat.xls";
if(defined $opts{p}){
print STAT "Progrom,Parameters,Input and Output information:\n";
print STAT "$0 adp_qual -f $file2 -r $file3 -c $qCutOff -q $format -a $adapter -l $lenCut -p -o $file4\n";
print STAT "Input file:$file2 and $file3\n";
print STAT "Low quality threshold:$qCutOff\n";
print STAT "Input file format:$format\n";
print STAT "Adapter sequence: $adapter\n";
print STAT "Output file: $file4"."_1.fastq and $file4"."_2.fastq\n";
print STAT "Output stat file: $file4.stat.xls\n\n";
}else{
print STAT "$0 adp_qual -i $file1 -c $qCutOff -q $format -a $adapter -l $lenCut -o $file4\n";
print STAT "Input file:$file1\n";
print STAT "Low quality threshold:$qCutOff\n";
print STAT "Input file format:$format\n";
print STAT "Adapter sequence: $adapter\n";
print STAT "Output file: $file4.fastq\n";
print STAT "Output stat file: $file4.stat.xls\n\n";
}
if(defined $opts{p}){ #Paired-End Format
open IN1,"$file2" || die $!;
open IN2,"$file3" || die $!;
open OUT1,">$file4"."_1.fastq" || die $!;
open OUT2,">$file4"."_2.fastq" || die $!;
open OUT3,">$file4"."_single_1.fastq" || die $!;
open OUT4,">$file4"."_single_2.fastq" || die $!;
while(!eof(IN1) && !eof(IN2)){
$id1 = <IN1>; $seq1 = <IN1>; <IN1>; $qual1 = <IN1>; $input++;
$id2 = <IN2>; $seq2 = <IN2>; <IN2>; $qual2 = <IN2>;
chomp ($id1,$seq1,$qual1,$id2,$seq2,$qual2);
$length1 = length $seq1;
$length2 = length $seq2;
$total_length1 = $total_length1 + $length1;
$total_length2 = $total_length2 + $length2;
##Remove adapter:
if($seq1 =~ m/$adapter/ && $seq2 =~ m/$adapter/){
$pos1 = index($seq1,$adapter);
$pos2 = index($seq2,$adapter);
if($pos1 < $lenCut or $pos2 < $lenCut){
$adp++;
next;
}else{
$seq1 = substr($seq1,0,$pos1 - 2);
$qual1 = substr($qual1,0,$pos1 - 2);
$seq2 = substr($seq2,0,$pos2 - 2);
$qual2 = substr($qual2,0,$pos2 - 2);
}
}elsif($seq1 =~ m/$adapter/){
$pos1 = index($seq1,$adapter);
if($pos1 < $lenCut){
$adp++;
next;
}else{
$seq1 = substr($seq1,0,$pos1 - 2);
$qual1 = substr($qual1,0,$pos1 - 2);
}
}elsif($seq2 =~ m/$adapter/){
$pos2 = index($seq2,$adapter);
if($pos2 < $lenCut){
$adp++;
next;
}else{
$seq2 = substr($seq2,0,$pos2 - 2);
$qual2 = substr($qual2,0,$pos2 - 2);
}
}
##Remove lower bases:
@qual1 = split (//, $qual1); @qual2 = split (//, $qual2);
$trimCount1 = $trimCount2 = $trimStart1 = $trimStart2 = 0;
for(my $i=$#qual1; $i>0; $i--) {
my $val1 = ord($qual1[$i]) - $format;
if($val1 < $qCutOff) {
$trimCount1++;
}else{
last;
}
}
for(my $i = 0;$i < $#qual1; $i++) {
my $val1 = ord($qual1[$i]) - $format;
if($val1 < $qCutOff) {
$trimStart1++;
}else{
last;
}
}
for(my $i=$#qual2; $i>0; $i--) {
my $val2 = ord($qual2[$i]) - $format;
if($val2 < $qCutOff) {
$trimCount2++;
}else{
last;
}
}
for(my $i = 0;$i < $#qual2; $i++) {
my $val2 = ord($qual2[$i]) - $format;
if($val2 < $qCutOff) {
$trimStart2++;
}else{
last;
}
}
$size1 = $length1 - $trimStart1 - $trimCount1;
$qual1 = substr($qual1,$trimStart1,$size1);
$seq1 = substr($seq1,$trimStart1,$size1);
$size2 = $length2 - $trimStart2 - $trimCount2;
$qual2 = substr($qual2,$trimStart2,$size2);
$seq2 = substr($seq2,$trimStart2,$size2);
$len1 = length $seq1;
$len2 = length $seq2;
if(($len1 >= $lenCut) && ($len2 >= $lenCut)){
print OUT1 "$id1\n$seq1\n+\n$qual1\n";
print OUT2 "$id2\n$seq2\n+\n$qual2\n";
$total_length_trimmed1 = $total_length_trimmed1 + $len1;
$total_length_trimmed2 = $total_length_trimmed2 + $len2;
$high1++; $high2++;
}elsif($len1 >= $lenCut){
print OUT3 "$id1\n$seq1\n+\n$qual1\n";
$high3++;$single_length1 = $single_length1 + $len1;
}elsif($len2 >= $lenCut){
print OUT4 "$id2\n$seq2\n+\n$qual2\n";
$high4++;$single_length2 = $single_length2 + $len2;
}
}
close IN1;
close IN2;
close OUT1;
close OUT2;
close OUT3;
close OUT4;
}else{ #Single Format
open IN1,"$file1" || die $!;
open OUT1,">$file4.fastq" || die $!;
while(!eof(IN1)){
$id1 = <IN1>; $seq1 = <IN1>; <IN1>; $qual1 = <IN1>; $input++;
chomp ($id1,$seq1,$qual1);
$length1 = length $seq1;
$total_length1 = $total_length1 + $length1;
#Remove adapter:
if($seq1 =~ m/$adapter/){
$pos = index($seq1,$adapter);
if($pos < $lenCut){
$adp++;
next;
}else{
$seq1 = substr($seq1,0,$pos - 2);
$qual1 = substr($qual1,0,$pos - 2);
}
}
##Remove lower bases:
@qual1 = split (//, $qual1);
$trimCount1 = $trimStart1 = 0;
for(my $i=$#qual1; $i>0; $i--) {
my $val = ord($qual1[$i]) - $format;
if($val < $qCutOff) {
$trimCount1++;
}else{
last;
}
}
for(my $i = 0;$i < $#qual1; $i++) {
my $val = ord($qual1[$i]) - $format;
if($val < $qCutOff) {
$trimStart1++;
}else{
last;
}
}
$size1 = $length1 - $trimStart1 - $trimCount1;
$qual1 = substr($qual1,$trimStart1,$size1);
$seq1 = substr($seq1,$trimStart1,$size1);
$len1 = length $seq1;
if($len1 >= $lenCut){
print OUT1 "$id1\n$seq1\n+\n$qual1\n";
$total_length_trimmed1 = $total_length_trimmed1 + $len1;
$high1++;
}
}
close IN1;
close OUT1;
}
if(defined $opts{p}){
my $rata_trimmed1 = int(10000*$total_length_trimmed1/$total_length1)/100;
my $rata_trimmed2 = int(10000*$total_length_trimmed2/$total_length2)/100;
my $single1 = int(10000*$single_length1/$total_length1)/100;
my $single2 = int(10000*$single_length2/$total_length2)/100;
my $reads = $input - $adp;
my $percentage = int(10000*$reads/$input)/100;
print STAT "Remove adpter information:\n";
print STAT "Name\tTotal_Reads\tValid_Data\tPercentage\n";
print STAT "$file2\t$input\t$reads\t$percentage%\n";
print STAT "$file3\t$input\t$reads\t$percentage%\n\n";
print STAT "Remove lower bases information:\n";
print STAT "Name\tTotal_Reads\tTotal_bases\tHigh_quility_reads\tHigh_quility_bases\tSingle_Reads\tSingle_bases\n";
print STAT "$file2\t$input\t$total_length1\t$high1\t$total_length_trimmed1($rata_trimmed1%)\t$high3\t$single_length1($single1%)\n";
print STAT "$file3\t$input\t$total_length2\t$high2\t$total_length_trimmed2($rata_trimmed2%)\t$high4\t$single_length2($single2%)\n\n";
}else{
my $rata_trimmed1 = int(10000*$total_length_trimmed1/$total_length1)/100;
my $reads = $input - $adp;
my $percentage = int(10000*$reads/$input)/100;
print STAT "Remove adpter information:\n";
print STAT "Name\tTotal_Reads\tValid_Data\tPercentage\n";
print STAT "$file1\t$input\t$reads\t$percentage%\n\n";
print STAT "Remove lower bases information:\n";
print STAT "Total reads: $input\n";
print STAT "Total bases: $total_length1\n";
print STAT "High quility reads: $high1\n";
print STAT "Total bases after trimming: $total_length_trimmed1($rata_trimmed1%)\n\n";
}
my $oo = time() - $oldtime;
$oo = int(100*$oo/60)/100;
print STAT "All completed successfully!\n\nTime consumption is $oo min";
close STAT;
}
#########################adp_dup#########################################
sub adp_dup {
open STAT,">$file4.stat.xls";
if(defined $opts{p}){
print STAT "Progrom,Parameters,Input and Output information:\n";
print STAT "$0 adp_dup -f $file2 -r $file3 -a $adapter -l $lenCut -p -o $file4\n";
print STAT "Input file:$file2 and $file3\n";
print STAT "Adapter sequence: $adapter\n";
print STAT "Output file: $file4"."_1.fastq and $file4"."_2.fastq\n";
print STAT "Output stat file: $file4.stat.xls\n\n";
}else{
print STAT "$0 adp_dup -i $file1 -a $adapter -l $lenCut -o $file4\n";
print STAT "Input file:$file1\n";
print STAT "Adapter sequence: $adapter\n";
print STAT "Output file: $file4.fastq\n";
print STAT "Output stat file: $file4.stat.xls\n\n";
}
if(defined $opts{p}){ #Paired-End Format
open IN1,"$file2" || die $!;
open IN2,"$file3" || die $!;
open OUT1,">$file4"."_1.fastq" || die $!;
open OUT2,">$file4"."_2.fastq" || die $!;
open OUT3,">$file4"."_single_1.fastq" || die $!;
open OUT4,">$file4"."_single_2.fastq" || die $!;
while(!eof(IN1) && !eof(IN2)){
$id1 = <IN1>; $seq1 = <IN1>; <IN1>; $qual1 = <IN1>; $input++;
$id2 = <IN2>; $seq2 = <IN2>; <IN2>; $qual2 = <IN2>;
chomp ($id1,$seq1,$qual1,$id2,$seq2,$qual2);
$length1 = length $seq1;
$length2 = length $seq2;
$total_length1 = $total_length1 + $length1;
$total_length2 = $total_length2 + $length2;
##Remove adapter:
if($seq1 =~ m/$adapter/ && $seq2 =~ m/$adapter/){
$pos1 = index($seq1,$adapter);
$pos2 = index($seq2,$adapter);
if($pos1 < $lenCut or $pos2 < $lenCut){
$adp++;
next;
}else{
$seq1 = substr($seq1,0,$pos1 - 2);
$qual1 = substr($qual1,0,$pos1 - 2);
$seq2 = substr($seq2,0,$pos2 - 2);
$qual2 = substr($qual2,0,$pos2 - 2);
}
}elsif($seq1 =~ m/$adapter/){
$pos1 = index($seq1,$adapter);
if($pos1 < $lenCut){
$adp++;
next;
}else{
$seq1 = substr($seq1,0,$pos1 - 2);
$qual1 = substr($qual1,0,$pos1 - 2);
}
}elsif($seq2 =~ m/$adapter/){
$pos2 = index($seq2,$adapter);
if($pos2 < $lenCut){
$adp++;
next;
}else{
$seq2 = substr($seq2,0,$pos2 - 2);
$qual2 = substr($qual2,0,$pos2 - 2);
}
}
$len1 = length $seq1;
$len2 = length $seq2;
if(($len1 >= $lenCut) && ($len2 >= $lenCut)){
##Remove duplication:
my $dup = substr($seq1,0,$lenCut).substr($seq2,0,$lenCut);
$hash{$dup}++;
if($hash{$dup} == 1){
print OUT1 "$id1\n$seq1\n+\n$qual1\n";
print OUT2 "$id2\n$seq2\n+\n$qual2\n";
$total_length_trimmed1 = $total_length_trimmed1 + $len1;
$total_length_trimmed2 = $total_length_trimmed2 + $len2;
$high1++; $high2++;
}else{
$dup_num++;
next;
}
}elsif($len1 >= $lenCut){
print OUT3 "$id1\n$seq1\n+\n$qual1\n";
$high3++;$single_length1 = $single_length1 + $len1;
}elsif($len2 >= $lenCut){
print OUT4 "$id2\n$seq2\n+\n$qual2\n";
$high4++;$single_length2 = $single_length2 + $len2;
}
}
close IN1;
close IN2;
close OUT1;
close OUT2;
close OUT3;
close OUT4;
}else{ #Single Format
open IN1,"$file1" || die $!;
open OUT1,">$file4.fastq" || die $!;
while(!eof(IN1)){
$id1 = <IN1>; $seq1 = <IN1>; <IN1>; $qual1 = <IN1>; $input++;
chomp ($id1,$seq1,$qual1);
$length1 = length $seq1;
$total_length1 = $total_length1 + $length1;
#Remove adapter:
if($seq1 =~ m/$adapter/){
$pos = index($seq1,$adapter);
if($pos < $lenCut){
$adp++;
next;
}else{
$seq1 = substr($seq1,0,$pos - 2);
$qual1 = substr($qual1,0,$pos - 2);
}
}
$len1 = length $seq1;
if($len1 >= $lenCut){
##Remove duplication:
my $dup = substr($seq1,0,$lenCut);
$hash{$dup}++;
if($hash{$dup} == 1){
print OUT1 "$id1\n$seq1\n+\n$qual1\n";
}else{
$dup_num++;
next;
}
}
}
close IN1;
close OUT1;
}
if(defined $opts{p}){
my $rata_trimmed1 = int(10000*$total_length_trimmed1/$total_length1)/100;
my $rata_trimmed2 = int(10000*$total_length_trimmed2/$total_length2)/100;
my $single1 = int(10000*$single_length1/$total_length1)/100;
my $single2 = int(10000*$single_length2/$total_length2)/100;
my $reads = $input - $adp;
my $percentage = int(10000*$reads/$input)/100;
my $dup_rate = int(10000*$dup_num/$reads)/100;
print STAT "Remove adpter information:\n";
print STAT "Name\tTotal_Reads\tValid_Data\tPercentage\n";
print STAT "$file2\t$input\t$reads\t$percentage%\n";
print STAT "$file3\t$input\t$reads\t$percentage%\n\n";
print STAT "Duplication information:\n";
print STAT "Total reads: $reads\n";
print STAT "Duplicate reads: $dup_num\n";
print STAT "Duplication: $dup_rate%\n\n";
}else{
my $rata_trimmed1 = int(10000*$total_length_trimmed1/$total_length1)/100;
my $reads = $input - $adp;
my $percentage = int(10000*$reads/$input)/100;
my $dup_rate = int(10000*$dup_num/$reads)/100;
print STAT "Remove adpter information:\n";
print STAT "Name\tTotal_Reads\tValid_Data\tPercentage\n";
print STAT "$file1\t$input\t$reads\t$percentage%\n\n";
print STAT "Duplication information:\n";
print STAT "Total reads: $reads\n";
print STAT "Duplicate reads: $dup_num\n";
print STAT "Duplication: $dup_rate%\n\n";
}
undef %hash;
my $oo = time() - $oldtime;
$oo = int(100*$oo/60)/100;
print STAT "All completed successfully!\n\nTime consumption is $oo min";
close STAT;
}
#########################all#########################################
sub all {
open STAT,">$file4.stat.xls";
if(defined $opts{p}){
print STAT "Progrom,Parameters,Input and Output information:\n";
print STAT "$0 all -f $file2 -r $file3 -c $qCutOff -q $format -a $adapter -l $lenCut -p -o $file4\n";
print STAT "Input file:$file2 and $file3\n";
print STAT "Low quality threshold:$qCutOff\n";
print STAT "Input file format:$format\n";
print STAT "Adapter sequence: $adapter\n";
print STAT "Output file: $file4.$qCutOff"."_1.fastq and $file4.$qCutOff"."_2.fastq\n";
print STAT "Output stat file: $file4.stat.xls\n\n";
}else{
print STAT "$0 all -i $file1 -c $qCutOff -q $format -a $adapter -l $lenCut -o $file4\n";
print STAT "Input file:$file1\n";
print STAT "Low quality threshold:$qCutOff\n";
print STAT "Input file format:$format\n";
print STAT "Adapter sequence: $adapter\n";
print STAT "Output file: $file4.$qCutOff.fastq\n";
print STAT "Output stat file: $file4.stat.xls\n\n";
}
if(defined $opts{p}){ #Paired-End Format
open IN1,"$file2" || die $!;
open IN2,"$file3" || die $!;
open OUT1,">$file4"."_1.fastq" || die $!;
open OUT2,">$file4"."_2.fastq" || die $!;
open OUT3,">$file4"."_single_1.fastq" || die $!;
open OUT4,">$file4"."_single_2.fastq" || die $!;
while(!eof(IN1) && !eof(IN2)){
$id1 = <IN1>; $seq1 = <IN1>; <IN1>; $qual1 = <IN1>; $input++;
$id2 = <IN2>; $seq2 = <IN2>; <IN2>; $qual2 = <IN2>;
chomp ($id1,$seq1,$qual1,$id2,$seq2,$qual2);
$length1 = length $seq1;
$length2 = length $seq2;
$total_length1 = $total_length1 + $length1;
$total_length2 = $total_length2 + $length2;
##Remove adapter:
if($seq1 =~ m/$adapter/ && $seq2 =~ m/$adapter/){
$pos1 = index($seq1,$adapter);
$pos2 = index($seq2,$adapter);
if($pos1 < $lenCut or $pos2 < $lenCut){
$adp++;
next;
}else{
$seq1 = substr($seq1,0,$pos1 - 2);
$qual1 = substr($qual1,0,$pos1 - 2);
$seq2 = substr($seq2,0,$pos2 - 2);
$qual2 = substr($qual2,0,$pos2 - 2);
}
}elsif($seq1 =~ m/$adapter/){
$pos1 = index($seq1,$adapter);
if($pos1 < $lenCut){
$adp++;
next;
}else{
$seq1 = substr($seq1,0,$pos1 - 2);
$qual1 = substr($qual1,0,$pos1 - 2);
}
}elsif($seq2 =~ m/$adapter/){
$pos2 = index($seq2,$adapter);
if($pos2 < $lenCut){
$adp++;
next;
}else{
$seq2 = substr($seq2,0,$pos2 - 2);
$qual2 = substr($qual2,0,$pos2 - 2);
}
}
##Remove lower bases:
@qual1 = split (//, $qual1); @qual2 = split (//, $qual2);
$trimCount1 = $trimCount2 = $trimStart1 = $trimStart2 = 0;
for(my $i=$#qual1; $i>0; $i--) {
my $val1 = ord($qual1[$i]) - $format;
if($val1 < $qCutOff) {
$trimCount1++;
}else{
last;
}
}
for(my $i = 0;$i < $#qual1; $i++) {
my $val1 = ord($qual1[$i]) - $format;
if($val1 < $qCutOff) {
$trimStart1++;
}else{
last;
}
}
for(my $i=$#qual2; $i>0; $i--) {
my $val2 = ord($qual2[$i]) - $format;
if($val2 < $qCutOff) {
$trimCount2++;
}else{
last;
}
}
for(my $i = 0;$i < $#qual2; $i++) {
my $val2 = ord($qual2[$i]) - $format;
if($val2 < $qCutOff) {
$trimStart2++;
}else{
last;
}
}
$size1 = $length1 - $trimStart1 - $trimCount1;
$qual1 = substr($qual1,$trimStart1,$size1);
$seq1 = substr($seq1,$trimStart1,$size1);
$size2 = $length2 - $trimStart2 - $trimCount2;
$qual2 = substr($qual2,$trimStart2,$size2);
$seq2 = substr($seq2,$trimStart2,$size2);
$len1 = length $seq1;
$len2 = length $seq2;
if(($len1 >= $lenCut) && ($len2 >= $lenCut)){
##Remove duplication:
my $dup = substr($seq1,0,$lenCut).substr($seq2,0,$lenCut);
$hash{$dup}++;
if($hash{$dup} == 1){
print OUT1 "$id1\n$seq1\n+\n$qual1\n";
print OUT2 "$id2\n$seq2\n+\n$qual2\n";
$total_length_trimmed1 = $total_length_trimmed1 + $len1;
$total_length_trimmed2 = $total_length_trimmed2 + $len2;