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trim_galore
executable file
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trim_galore
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#!/usr/bin/perl
use strict;
use warnings;
use Getopt::Long;
use IPC::Open3;
use File::Spec;
use File::Basename;
use Cwd;
## This program is Copyright (C) 2012-23, Felix Krueger (fkrueger@altoslabs.com)
## Edited by Frankie James (github.com/fjames003) for multi-core support
## This program is free software: you can redistribute it and/or modify
## it under the terms of the GNU General Public License as published by
## the Free Software Foundation, either 3 of the License, or
## (at your option) any later version.
## This program is distributed in the hope that it will be useful,
## but WITHOUT ANY WARRANTY; without even the implied warranty of
## MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the
## GNU General Public License for more details.
## You should have received a copy of the GNU General Public License
## along with this program. If not, see <http://www.gnu.org/licenses/>.
## This script is taking in FastQ sequences and trims them using Cutadapt
## modified on 15 01 2022 to add Illumina directional mRNA kit adapter (ACTGTCTCTTATACACATCT)
## modified on 12 Aug 2022 to allow --max_n to be a fraction
my $DOWARN = 1; # print on screen warning and text by default
BEGIN { $SIG{'__WARN__'} = sub { warn $_[0] if $DOWARN } };
my $trimmer_version = '0.6.10';
my $last_modified = "02 02 2023";
my $cutadapt_version;
my $python_version;
my ($compression_path,$decompression_path,$cores,$cutoff,$adapter,$stringency,$rrbs,$length_cutoff,$keep,$fastqc,$non_directional,$phred_encoding,$fastqc_args,$trim,$gzip,$validate,$retain,$length_read_1,$length_read_2,$a2,$error_rate,$output_dir,$no_report_file,$dont_gzip,$clip_r1,$clip_r2,$three_prime_clip_r1,$three_prime_clip_r2,$nextera,$stranded_illumina,$small_rna,$path_to_cutadapt,$illumina,$max_length,$maxn,$maxn_fraction,$trim_n,$hardtrim5,$clock,$polyA,$hardtrim3,$nextseq,$basename,$consider_already_trimmed,$umi_from_r2) = process_commandline();
my $report_message; # stores result of adapter auto-detection
my @filenames = @ARGV;
die "\nPlease provide the filename(s) of one or more FastQ file(s) to launch Trim Galore!\n
USAGE: 'trim_galore [options] <filename(s)>' or 'trim_galore --help' for more options\n\n" unless (@filenames);
file_sanity_check($filenames[0]);
################################################################
### ###
### SPECIALITY TRIMMING SECTION - EXITING AFTERWARDS ###
### ###
################################################################
if (defined $hardtrim5){
warn "Hard-trimming from the 3'-end selected. File(s) will be trimmed to leave the leftmost $hardtrim5 bp on the 5'-end, and Trim Galore will then exit.\n\n";
foreach my $file(@filenames){
hardtrim_to_5prime_end($file);
}
exit;
}
if (defined $hardtrim3){
warn "Hard-trimming from 5'-end selected. File(s) will be trimmed to leave the rightmost $hardtrim3 bp on the 3'-end, and Trim Galore will then exit.\n\n";
foreach my $file(@filenames){
hardtrim_to_3prime_end($file);
}
exit;
}
if ($clock){
warn "\nIT'S TIME FOR CLOCK PROCESSING!!!\t\t\t\t\t\t\t\t\t[pun intended]\n\n";
while (@filenames){
my $in1 = shift @filenames;
my $in2 = shift @filenames;
clockwork($in1,$in2);
}
warn "\nPre-processing finished...\n\nPlease run Trim Galore again to remove adapters, poor quality bases as well as UMI/fixed sequences from the 3'-end of the reads.\nA sample command for this is:\n\n~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~\ntrim_galore --paired --three_prime_clip_R1 15 --three_prime_clip_R2 15 *.clock_UMI.R1.fq.gz *.clock_UMI.R2.fq.gz\n~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~\n\nTrim Galore Epigenetic Clock processing complete.\n\n";
exit;
}
if (defined $umi_from_r2){
my $umi_length = $umi_from_r2;
warn "~"x120 . "\n";
warn "\nTrim Galore speciality trimming: transferring the first $umi_length bp from Read 2 to the read ID (of both reads)\n\n";
warn "~"x120 . "\n\n";
while (@filenames){
my $in1 = shift @filenames;
my $in2 = shift @filenames;
implicon_umi($in1,$in2,$umi_length);
}
warn "~"x120 . "\n";
warn "\nPre-processing finished. ${umi_length}bp from the 5'-end of READ2 have been transferred to the Read ID of both R1 and R2. Exiting...\n\n";
warn "~"x120 . "\n\n";
exit;
}
sub implicon_umi{
### FILEHANDLES
my ($in1_fh,$in2_fh); # input filehandles
my ($read1_fh,$read2_fh); # output filehandles
### INPUT FILES
my ($in1,$in2,$umi_length) = @_;
if ($in1 =~ /\.gz$/){
open ($in1_fh,"$decompression_path -d -c $in1 |") or die "Failed to read from file $in1: $!";
}
else{
open ($in1_fh,$in1) or die "Failed to read from file $in1: $!";
}
if ($in2 =~ /\.gz$/){
open ($in2_fh,"$decompression_path -d -c $in2 |") or die "Failed to read from file $in2: $!";
}
else{
open ($in2_fh,$in2) or die "Failed to read from file $in2: $!";
}
# warn " Input file name 1: $in1\n";
# warn " Input file name 2: $in2\n";
### OUTPUT FILES
my $out1 = (split (/\//,$in1))[-1];
my $out2 = (split (/\//,$in2))[-1];
$out1 =~ s/(\.fastq$|\.fastq\.gz$)//;
$out1 =~ s/(\.fq$|\.fq\.gz$)//;
$out1 =~ s/_?R1$//;
$out1 .= "_${umi_length}bp_UMI_R1.fastq"; # appending to the end
$out2 =~ s/(\.fastq$|\.fastq\.gz$)//;
$out2 =~ s/(\.fq$|\.fq\.gz$)//;
$out2 =~ s/_?R[234]$//;
$out2 .= "_${umi_length}bp_UMI_R2.fastq"; # appending to the end
### READ 1
if ($gzip or $in1 =~ /\.gz$/){
if ($dont_gzip){
open ($read1_fh,'>',$output_dir.$out1) or die "Can't open '$out1': $!\n";
}
else{
$out1 .= '.gz';
open ($read1_fh,"| $compression_path -c - > ${output_dir}${out1}") or die "Can't write to '$out1': $!\n";
}
}
else{
open ($read1_fh,'>',$output_dir.$out1) or die "Can't open '$out1': $!\n";
}
warn "Writing UMI trimmed version of the input file '$in1' to '$out1'\n";
### READ 2
if ($gzip or $in2 =~ /\.gz$/){
if ($dont_gzip){
open ($read2_fh,'>',$output_dir.$out2) or die "Can't open '$out2': $!\n";
}
else{
$out2 .= '.gz';
open ($read2_fh,"| $compression_path -c - > ${output_dir}${out2}") or die "Can't write to '$out2': $!\n";
}
}
else{
open ($read2_fh,'>',$output_dir.$out2) or die "Can't open '$out2': $!\n";
}
warn "Writing UMI trimmed version of the input file '$in2' to '$out2'\n\n";
my %freqs;
my $umi_1;
my $umi_2;
# print "Processing files $in1 and $in2\n";
my %r1; # storing the barcodes for R1
my %r2; # storing the barcodes for R2
my $count = 0;
TYRION: while (1){
my $one1 = <$in1_fh>;
my $one2 = <$in1_fh>;
my $one3 = <$in1_fh>;
my $one4 = <$in1_fh>;
my $two1 = <$in2_fh>;
my $two2 = <$in2_fh>;
my $two3 = <$in2_fh>;
my $two4 = <$in2_fh>;
last unless ($one4 and $two4);
# chomp $one2; # sequence. currently not required
chomp $two2; # sequence
chomp $one1; # read ID, need to append UMIs to the read ID
chomp $two1; # read ID, need to append UMIs to the read ID
# chomp $one4; # quality. currently not required
chomp $two4; # quality
++$count; # sequence count
if ($count % 1000000 ==0){
warn "Processed $count sequences so far...\n";
}
### For IMPLICON SEQUNCING, READ2 looks like this:
# 0 8 INDEX position
# UUUUUUUU FFFFFFFFFFFFFFFFF....
# where:
# U = UMI base
# F = SEQUENCE to be aligned
### Capturing the sequence after the UMI (FFFFFFFFFFFFFF...)
my $r2_barcode = substr($two2,0,$umi_length);
my $seq2 = substr($two2,$umi_length); # truncated sequence without UMIs
my $qual2 = substr($two4,$umi_length); # truncated quality string without barcode
# warn "$r2_barcode\n$two2\n$two4\n$seq2\n$qual2\n"; sleep(1);
# APPENDING TO THE READ IDs
# warn "$one1\n";
$one1 .= ":${r2_barcode}";
# warn "$one1\n";
#warn "$two1\n";
$two1 .= ":${r2_barcode}";
# warn "$two1\n";
# warn "=====\n$one2\n$one2\n$one4\n$one4\n~~\n$two2\n $seq2\n$two4\n$qual2\n=====\n\n";sleep(1);
print ${read1_fh} "$one1\n";
print ${read1_fh} "$one2";
print ${read1_fh} "+\n"; # replacing this with a + for space and format reasons
print ${read1_fh} "$one4";
print ${read2_fh} "$two1\n";
print ${read2_fh} "$seq2\n";
print ${read2_fh} "+\n"; # replacing this with a + for space and format reasons
print ${read2_fh} "$qual2\n";
# $r1{$r1_barcode}++;
# $r2{$r2_barcode}++;
}
warn "Sequences processed in total: $count\n\n ~~~~~~~~~~~~~~~~~~~~~~~~~~~\n\n";
}
sub clockwork{
### FILEHANDLES
my ($in1_fh,$in2_fh); # input filehandles
my ($read1_fh,$read2_fh); # output filehandles
### INPUT FILES
my ($in1,$in2) = @_;
if ($in1 =~ /\.gz$/){
open ($in1_fh,"$decompression_path -d -c $in1 |") or die "Failed to read from file $in1: $!";
}
else{
open ($in1_fh,$in1) or die "Failed to read from file $in1: $!";
}
if ($in2 =~ /\.gz$/){
open ($in2_fh,"$decompression_path -d -c $in2 |") or die "Failed to read from file $in2: $!";
}
else{
open ($in2_fh,$in2) or die "Failed to read from file $in2: $!";
}
# warn " Input file name 1: $in1\n";
# warn " Input file name 2: $in2\n";
### OUTPUT FILES
my $out1 = (split (/\//,$in1))[-1];
my $out2 = (split (/\//,$in2))[-1];
$out1 =~ s/(\.fastq$|\.fastq\.gz$)//;
$out1 =~ s/(\.fq$|\.fq\.gz$)//;
$out1 .= '.clock_UMI.R1.fq'; # appending to the end
$out2 =~ s/(\.fastq$|\.fastq\.gz$)//;
$out2 =~ s/(\.fq$|\.fq\.gz$)//;
$out2 .= '.clock_UMI.R2.fq'; # appending to the end
### READ 1
if ($gzip or $in1 =~ /\.gz$/){
if ($dont_gzip){
open ($read1_fh,'>',$output_dir.$out1) or die "Can't open '$out1': $!\n";
}
else{
$out1 .= '.gz';
open ($read1_fh,"| $compression_path -c - > ${output_dir}${out1}") or die "Can't write to '$out1': $!\n";
}
}
else{
open ($read1_fh,'>',$output_dir.$out1) or die "Can't open '$out1': $!\n";
}
warn "Writing dual trimmed version of the input file '$in1' to '$out1'\n";
### READ 2
if ($gzip or $in2 =~ /\.gz$/){
if ($dont_gzip){
open ($read2_fh,'>',$output_dir.$out2) or die "Can't open '$out2': $!\n";
}
else{
$out2 .= '.gz';
open ($read2_fh,"| $compression_path -c - > ${output_dir}${out2}") or die "Can't write to '$out2': $!\n";
}
}
else{
open ($read2_fh,'>',$output_dir.$out2) or die "Can't open '$out2': $!\n";
}
warn "Writing dual trimmed version of the input file '$in2' to '$out2'\n ---\n";
# warn "Output file name 1: $out1\n";
# warn "Output file name 2: $out2\n";
my %freqs;
my $umi_1;
my $umi_2;
# open ($out2,"| $compression_path -c - > $out1") or die $!;
# open (OUT2,"| $compression_path -c - > $out2") or die $!;
# print "Processing files $in1 and $in2\n";
my %r1; # storing the barcodes for R1
my %r2; # storing the barcodes for R2
my %fix1; # storing the fixed sequence (CAGT + A from A-tailing) of R1
my %fix2; # storing the fixed sequence (CAGT + A from A-tailing) of R2
my $count = 0;
my $filtered_count = 0;
my $r1_contains_rc = 0;
ORANGE: while (1){
my $one1 = <$in1_fh>;
my $one2 = <$in1_fh>;
my $one3 = <$in1_fh>;
my $one4 = <$in1_fh>;
my $two1 = <$in2_fh>;
my $two2 = <$in2_fh>;
my $two3 = <$in2_fh>;
my $two4 = <$in2_fh>;
last unless ($one4 and $two4);
chomp $one2; # sequence
chomp $two2; # sequence
chomp $one1; # read ID, need to append UMIs to the read ID
chomp $two1; # read ID, need to append UMIs to the read ID
chomp $one4; # quality
chomp $two4; # quality
++$count; # sequence count
if ($count % 1000000 ==0){
warn "Processed $count sequences so far...\n";
}
my $r1_barcode;
my $r2_barcode;
my $r1_fix;
my $r2_fix;
$r1_barcode = substr($one2,0,8);
$r2_barcode = substr($two2,0,8);
$r1_fix = substr($one2,8,4);
$r2_fix = substr($two2,8,4);
# warn "$one2\n$two2\n$r1_barcode\t$r1_fix\n$r2_barcode\t$r2_fix\n\n";sleep(1);
# this part of code simply counts the different types of sequence that occurred at the constant region where we expected
# to read CAGT in both reads. Both R1 and R2 are taken into account at the same time
unless ($r1_fix eq 'CAGT' and $r2_fix eq 'CAGT'){
$freqs{$r1_fix}++;
$freqs{$r2_fix}++;
$filtered_count++;
}
### BOTH READ1 AND READ2 should look like this:
# 0 8 12 13 INDEX position
# UUUUUUUU CAGT A FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF SEQUENCE
# where:
# U = UMI base
# CAGT = fixed sequence
# A = A-tail
# FFFF = RRBS-fragment to be aligned
### Capturing the sequence after the A from A-tailing (FFFFFFFFFFFFFF...)
my $seq1 = substr($one2,13); # truncated sequence without UMIs or fixed sequence
my $seq2 = substr($two2,13);
my $qual1 = substr($one4,13); # truncated quality string without barcode or fixed sequence
my $qual2 = substr($two4,13);
# warn "$one1\n";
$one1 .= ":R1:${r1_barcode}:R2:${r2_barcode}:F1:${r1_fix}:F2:${r2_fix}";
#warn "$one1\n";
#warn "$two1\n";
$two1 .= ":R1:${r1_barcode}:R2:${r2_barcode}:F1:${r1_fix}:F2:${r2_fix}";
#warn "$two1\n";
# warn "$one2\n $seq1\n$one4\n $qual1\n~~\n$two2\n $seq2\n$two4\n $qual2\n\n";sleep(1);
print ${read1_fh} "$one1\n";
print ${read1_fh} "$seq1\n";
print ${read1_fh} "+\n"; # replacing this with a + for space and format reasons
print ${read1_fh} "$qual1\n";
print ${read2_fh} "$two1\n";
print ${read2_fh} "$seq2\n";
print ${read2_fh} "+\n"; # replacing this with a + for space and format reasons
print ${read2_fh} "$qual2\n";
# sleep(1);
$r1{$r1_barcode}++;
$r2{$r2_barcode}++;
$fix1{$r1_fix}++;
$fix2{$r2_fix}++;
}
my $perc;
if ($count){
$perc = sprintf("%.2f",$filtered_count/$count * 100);
}
else{
$perc = 'N/A';
}
warn "Sequences processed in total: $count\nthereof had fixed sequence CAGT in both R1 and R2:\t $filtered_count ($perc%)\n ~~~~~~~~~~~~~~~~~~~~~~~~~~~\n\n";
}
################################################################
### ###
### SPECIALITY TRIMMING SECTION - END ###
### ###
################################################################
########################################################################
my $path_to_fastqc = 'fastqc';
########################################################################
### SETTING DEFAULTS UNLESS THEY WERE SPECIFIED
unless (defined $cutoff){
unless ($nextseq){
$cutoff = 20;
}
}
my $phred_score_cutoff = $cutoff; # only relevant for report
my $adapter_name = '';
unless (defined $adapter){
if ($nextera){
$adapter = 'CTGTCTCTTATA';
$adapter_name = 'Nextera Transposase sequence; user defined';
}
elsif($small_rna){
$adapter = 'TGGAATTCTCGG';
$adapter_name = 'Illumina small RNA adapter; user defined';
}
elsif($illumina){
$adapter = 'AGATCGGAAGAGC';
$adapter_name = 'Illumina TruSeq, Sanger iPCR; user defined';
}
elsif($stranded_illumina){
# more information available here:
# "Trimming T-overhang options for the Illumina Stranded mRNA and Illumina Stranded Total RNA workflows"
# https://support.illumina.com/bulletins/2020/06/trimming-t-overhang-options-for-the-illumina-rna-library-prep-wo.html
$adapter = 'ACTGTCTCTTATA';
$adapter_name = 'Illumina stranded mRNA and Total RNA; user defined (added for v0.6.8)';
}
else{ # default
### If other -a and/or -a2 were given
if ($polyA){ # specialised PolyA trimming
($adapter,$adapter_name) = autodetect_polyA_type();
if ($validate){ # we need to select -a2 as the reverse complement to -a
if ($adapter =~ /A+/){
$adapter = extend_adapter_sequence("A",20); # defaulting to 20
$a2 = extend_adapter_sequence("T",150); # defaulting to 150 bp
}
elsif($adapter =~ /T+/){
$adapter = extend_adapter_sequence("T",20); # defaulting to 20
$a2 = extend_adapter_sequence("A",150);
}
else{
die "Something unexpected happened with the Poly-A autodetection, please check\n";
}
# warn "Paired-end Poly-A detection, set the following parameters:\n";
# warn " -a: $adapter\n-a2: $a2\n ~~~~~~~~~~ \n\n";sleep(1);
}
else{
# Single end
if ($adapter =~ /A+/){
$adapter = extend_adapter_sequence("A",20); # defaulting to 20
}
elsif($adapter =~ /T+/){
$adapter = extend_adapter_sequence("T",20); # defaulting to 20
}
else{
die "Something unexpected happened with the Poly-A autodetection, please check\n";
}
}
}
else{ # ADAPTER TRIMMING; DEFAULT
($adapter,$adapter_name,$report_message) = autodetect_adapter_type();
}
}
}
else{
$adapter_name = 'user defined';
}
### For smallRNA adapters we are reducing the sequence length cutoff before a sequences gets thrown out entirely to 18bp. We are doing this because some 20-23bp long smallRNAs
### may be removed if T, TG, or TGG etc gets trimmed off the end (changed b ack up from 16 to 18bp to remove noise from alignment files, 18 11 2015)
if ($adapter eq 'TGGAATTCTCGG'){
unless (defined $length_cutoff){ # user defined length cutoff wins over auto-detection
$length_cutoff = 18;
warn "Reducing length cutoff to 18bp for small RNA-Seq reads because a cutoff of 20bp may remove some short species of small RNAs if they had been trimmed by 1,2 or 3bp\n";
}
### If the file is a smallRNA library and paired-end we set the Illumina 5' adapter as the $a2 sequence
if ($validate){
unless (defined $a2){
$a2 = 'GATCGTCGGACT';
warn "Setting the Illumina smallRNA 5' adapter as adapter 2: 'GATCGTCGGACT'\n";
}
}
}
unless (defined $length_cutoff){
$length_cutoff = 20; # non small RNA length cutoff
}
unless (defined $a2){ # optional adapter for the second read in a pair. Only works for --paired trimming
$a2 = '';
}
unless (defined $stringency){
$stringency = 1;
}
if ($phred_encoding == 64){
$cutoff += 31;
}
my $file_1;
my $file_2;
foreach my $filename (@ARGV){
trim ($filename);
}
sub trim{
my $filename = shift;
my $output_filename = (split (/\//,$filename))[-1];
my $report = $output_filename;
$report =~ s/$/_trimming_report.txt/;
if ($no_report_file) {
$report = File::Spec->devnull;
open (REPORT,'>',$report) or die "Failed to write to file '$report': $!\n";
# warn "Redirecting report output to /dev/null\n";
}
else{
open (REPORT,'>',$output_dir.$report) or die "Failed to write to file '$report': $!\n";
warn "Writing report to '$output_dir$report'\n";
}
warn "\nSUMMARISING RUN PARAMETERS\n==========================\nInput filename: $filename\n";
print REPORT "\nSUMMARISING RUN PARAMETERS\n==========================\nInput filename: $filename\n";
if ($validate){ # paired-end mode
warn "Trimming mode: paired-end\n";
print REPORT "Trimming mode: paired-end\n";
}
else{
warn "Trimming mode: single-end\n";
print REPORT "Trimming mode: single-end\n";
}
warn "Trim Galore version: $trimmer_version\n";
print REPORT "Trim Galore version: $trimmer_version\n";
warn "Cutadapt version: $cutadapt_version\n";
print REPORT "Cutadapt version: $cutadapt_version\n";
if (defined $python_version){
warn "Python version: $python_version\n";
print REPORT "Python version: $python_version\n";
}
if (defined $cores){
my $temp = $cores;
$temp =~ s/-j //;
warn "Number of cores used for trimming: $temp\n";
print REPORT "Number of cores used for trimming: $temp\n";
}
if (defined $phred_score_cutoff){
warn "Quality Phred score cutoff: $phred_score_cutoff\n";
print REPORT "Quality Phred score cutoff: $phred_score_cutoff\n";
}
warn "Quality encoding type selected: ASCII+$phred_encoding\n";
print REPORT "Quality encoding type selected: ASCII+$phred_encoding\n";
if ($report_message){
# warn "adding Auto-detection statement\n";
print REPORT $report_message;
}
warn "Adapter sequence: '$adapter' ($adapter_name)\n";
print REPORT "Adapter sequence: '$adapter' ($adapter_name)\n";
if ($error_rate == 0.1){
warn "Maximum trimming error rate: $error_rate (default)\n";
}
else{
warn "Maximum trimming error rate: $error_rate\n";
}
if (defined $maxn){
if (defined $maxn_fraction){
warn "Fraction of tolerated Ns: $maxn_fraction\n";
}
else{
warn "Maximum number of tolerated Ns: $maxn\n";
}
}
if ($nextseq){
warn "2-colour high quality G-trimming enabled, with quality cutoff: $nextseq\n";
print REPORT "2-colour high quality G-trimming enabled, with quality cutoff: $nextseq\n";
}
print REPORT "Maximum trimming error rate: $error_rate";
if ($error_rate == 0.1){
print REPORT " (default)\n";
}
else{
print REPORT "\n";
}
if ($a2){
warn "Optional adapter 2 sequence (only used for read 2 of paired-end files): '$a2'\n";
print REPORT "Optional adapter 2 sequence (only used for read 2 of paired-end files): '$a2'\n";
}
warn "Minimum required adapter overlap (stringency): $stringency bp\n";
print REPORT "Minimum required adapter overlap (stringency): $stringency bp\n";
if (defined $consider_already_trimmed){
warn "During adapter auto-detection, files are considered already adapter-trimmed if the highest found adapter was found equal to or lower than: $consider_already_trimmed\n";
print REPORT "During adapter auto-detection, files are considered already adapter-trimmed if the highest found adapter was found equal to or lower than: $consider_already_trimmed\n";
}
if ($validate){
warn "Minimum required sequence length for both reads before a sequence pair gets removed: $length_cutoff bp\n";
print REPORT "Minimum required sequence length for both reads before a sequence pair gets removed: $length_cutoff bp\n";
}
else{
warn "Minimum required sequence length before a sequence gets removed: $length_cutoff bp\n";
print REPORT "Minimum required sequence length before a sequence gets removed: $length_cutoff bp\n";
}
if ($max_length){
warn "Maxiumum tolerated read length after trimming (for smallRNA trimming): $max_length bp\n";
print REPORT "Maxiumum tolerated read length after trimming (for smallRNA trimming): $max_length bp\n";
}
if ($trim_n){
warn "Removing Ns from the start and end of reads\n";
print REPORT "Removing Ns from the start and end of reads\n";
}
if ($validate){ # only for paired-end files
if ($retain){ # keeping single-end reads if only one end is long enough
if ($length_read_1 == 35){
warn "Length cut-off for read 1: $length_read_1 bp (default)\n";
print REPORT "Length cut-off for read 1: $length_read_1 bp (default)\n";
}
else{
warn "Length cut-off for read 1: $length_read_1 bp\n";
print REPORT "Length cut-off for read 1: $length_read_1 bp\n";
}
if ($length_read_2 == 35){
warn "Length cut-off for read 2: $length_read_2 bb (default)\n";
print REPORT "Length cut-off for read 2: $length_read_2 bp (default)\n";
}
else{
warn "Length cut-off for read 2: $length_read_2 bp\n";
print REPORT "Length cut-off for read 2: $length_read_2 bp\n";
}
}
}
if ($rrbs){
warn "File was specified to be an MspI-digested RRBS sample. Read 1 sequences with adapter contamination will be trimmed a further 2 bp from their 3' end, and Read 2 sequences will be trimmed by 2 bp from their 5' end to remove potential methylation-biased bases from the end-repair reaction\n";
print REPORT "File was specified to be an MspI-digested RRBS sample. Read 1 sequences with adapter contamination will be trimmed a further 2 bp from their 3' end, and Read 2 sequences will be trimmed by 2 bp from their 5' end to remove potential methylation-biased bases from the end-repair reaction\n";
}
if ($non_directional){
warn "File was specified to be a non-directional MspI-digested RRBS sample. Sequences starting with either 'CAA' or 'CGA' will have the first 2 bp trimmed off to remove potential methylation-biased bases from the end-repair reaction\n";
print REPORT "File was specified to be a non-directional MspI-digested RRBS sample. Sequences starting with either 'CAA' or 'CGA' will have the first 2 bp trimmed off to remove potential methylation-biased bases from the end-repair reaction\n";
}
if ($clip_r1){
warn "All Read 1 sequences will be trimmed by $clip_r1 bp from their 5' end to avoid poor qualities or biases\n";
print REPORT "All Read 1 sequences will be trimmed by $clip_r1 bp from their 5' end to avoid poor qualities or biases\n";
}
if ($clip_r2){
warn "All Read 2 sequences will be trimmed by $clip_r2 bp from their 5' end to avoid poor qualities or biases (e.g. M-bias for BS-Seq applications)\n";
print REPORT "All Read 2 sequences will be trimmed by $clip_r2 bp from their 5' end to avoid poor qualities or biases (e.g. M-bias for BS-Seq applications)\n";
}
if ($three_prime_clip_r1){
warn "All Read 1 sequences will be trimmed by $three_prime_clip_r1 bp from their 3' end to avoid poor qualities or biases\n";
print REPORT "All Read 1 sequences will be trimmed by $three_prime_clip_r1 bp from their 3' end to avoid poor qualities or biases\n";
}
if ($three_prime_clip_r2){
warn "All Read 2 sequences will be trimmed by $three_prime_clip_r2 bp from their 3' end to avoid poor qualities or biases\n";
print REPORT "All Read 2 sequences will be trimmed by $three_prime_clip_r2 bp from their 3' end to avoid poor qualities or biases\n";
}
if ($fastqc){
warn "Running FastQC on the data once trimming has completed\n";
print REPORT "Running FastQC on the data once trimming has completed\n";
if ($fastqc_args){
warn "Running FastQC with the following extra arguments: '$fastqc_args'\n";
print REPORT "Running FastQC with the following extra arguments: $fastqc_args\n";
}
}
if ($keep and $rrbs){
warn "Keeping quality trimmed (but not yet adapter trimmed) intermediate FastQ file\n";
print REPORT "Keeping quality trimmed (but not yet adapter trimmed) intermediate FastQ file\n";
}
if ($gzip or $filename =~ /\.gz$/){
$gzip = 1;
unless ($dont_gzip){
warn "Output file(s) will be GZIP compressed\n";
print REPORT "Output file will be GZIP compressed\n";
}
}
warn "\n";
print REPORT "\n";
# sleep (3);
my $temp;
### We need to make sure that Cutadapt still runs if users use a fairly old version of Cutadapt, as multi-core handling is only supported sincce
### Cutadapt version 1.15. Edited on 08 03 2019
# if the Cutadapt version has more . in the version, we discard the second one.
# warn "Version was: $cutadapt_version\n";
# warn "Version now is: '$cutadapt_version'\n";
if ($cutadapt_version =~ /(\d+\.\d+).*/){
$cutadapt_version = $1;
}
#warn "Version now is: $cutadapt_version\n";
# sleep(5);
my ($major_version,$sub_version) = ($1,$2) if ($cutadapt_version =~ /^(\d+)\.(\d+)$/);
# warn "Major: $major_version\nSub version: $sub_version\n";
# sleep(3);
if ($major_version == 1){ # versions prior to 1.15 did not support the -j option
if ($sub_version < 15){
if ($cores > 1){
die "I'm sorry but your version of Cutadapt is too old to support multi-core trimming. Please update Cutadapt, and try again\n\n";
}
else{ #default single-core prcessing with old version of Cutadapt
warn "Your version of Cutadapt is fairly old (detected v$cutadapt_version), please consider updating Cutadapt!\n";
$cores = ""; # need to delete -j entirely as Cutadapt
}
}
else{
warn "Cutadapt seems to be reasonably up-to-date. Setting -j $cores\n";
unless ($cores =~ /^-j \d+$/){ # if it was set before, don't change it again
$cores = "-j $cores";
}
}
}
elsif ($major_version > 1){
warn "Cutadapt seems to be fairly up-to-date (version $cutadapt_version). Setting -j $cores\n";
unless ($cores =~ /^-j \d+$/){ # if it was set before, don't change it again
$cores = "-j $cores";
}
}
else{
die "Cutadapt major version was not 1 or higher. Simply too old...\n";
}
### Proceeding differently for RRBS and other type of libraries
if ($rrbs){
### Skipping quality filtering for RRBS libraries if a quality cutoff of 0 was specified
if ($cutoff == 0){
warn "Quality cutoff selected was 0 - Skipping quality trimming altogether\n\n";
# sleep (3);
}
else{
$temp = $filename;
$temp =~ s/^.*\///; # replacing optional file path information
$temp =~ s/$/_qual_trimmed.fastq/;
open (TEMP,'>',$output_dir.$temp) or die "Can't write to '$temp': $!";
warn " >>> Now performing adaptive quality trimming with a Phred-score cutoff of: $cutoff <<<\n\n";
# sleep (1);
open (QUAL,"$path_to_cutadapt $cores -e $error_rate -q $cutoff -a X $filename |") or die "Can't open pipe to Cutadapt: $!";
my $qual_count = 0;
while (1){
my $l1 = <QUAL>;
my $seq = <QUAL>;
my $l3 = <QUAL>;
my $qual = <QUAL>;
last unless (defined $qual);
$qual_count++;
if ($qual_count%10000000 == 0){
warn "$qual_count sequences processed\n";
}
print TEMP "$l1$seq$l3$qual";
}
warn "\n >>> Quality trimming completed <<<\n$qual_count sequences processed in total\n\n";
close QUAL or die "Unable to close QUAL filehandle: $!\n";
}
}
if ($output_filename =~ /\.fastq$/){
$output_filename =~ s/\.fastq$/_trimmed.fq/;
}
elsif ($output_filename =~ /\.fastq\.gz$/){
$output_filename =~ s/\.fastq\.gz$/_trimmed.fq/;
}
elsif ($output_filename =~ /\.fq$/){
$output_filename =~ s/\.fq$/_trimmed.fq/;
}
elsif ($output_filename =~ /\.fq\.gz$/){
$output_filename =~ s/\.fq\.gz$/_trimmed.fq/;
}
else{
$output_filename =~ s/$/_trimmed.fq/;
}
if ($gzip or $filename =~ /\.gz$/){
if ($dont_gzip){
open (OUT,'>',$output_dir.$output_filename) or die "Can't open '$output_filename': $!\n"; # don't need to gzip intermediate file
}
else{
### 6 Jan 2014: had a request to also gzip intermediate files to save disk space
# if ($validate){
# open (OUT,'>',$output_dir.$output_filename) or die "Can't open '$output_filename': $!\n"; # don't need to gzip intermediate file
# }
$output_filename .= '.gz';
open (OUT,"| $compression_path -c - > ${output_dir}${output_filename}") or die "Can't write to '$output_filename': $!\n";
}
}
else{
open (OUT,'>',$output_dir.$output_filename) or die "Can't open '$output_filename': $!\n";
}
warn "Writing final adapter and quality trimmed output to $output_filename\n\n";
my $count = 0;
my $too_short = 0;
my $too_long = 0;
my $too_many_n = 0;
my $quality_trimmed = 0;
my $rrbs_trimmed = 0;
my $rrbs_trimmed_start = 0;
my $CAA = 0;
my $CGA = 0;
my $pid;
if ($rrbs and $cutoff != 0){
### optionally using 2 different adapters for read 1 and read 2
if ($validate and $a2){
### Figure out whether current file counts as read 1 or read 2 of paired-end files
if ( scalar(@filenames)%2 == 0){ # this is read 1 of a pair
warn "\n >>> Now performing adapter trimming for the adapter sequence: '$adapter' from file $temp <<< \n";
#sleep (1);
$pid = open3 (\*WRITER, \*TRIM, \*ERROR,"$path_to_cutadapt $cores -e $error_rate -O $stringency -a $adapter $output_dir$temp") or die "Failed to launch Cutadapt: $!\n";
}
else{ # this is read 2 of a pair
warn "\n >>> Now performing adapter trimming for the adapter sequence: '$a2' from file $temp <<< \n";
#sleep (1);
$pid = open3 (\*WRITER, \*TRIM, \*ERROR,"$path_to_cutadapt $cores -e $error_rate -O $stringency -a $a2 $output_dir$temp") or die "Failed to launch Cutadapt: $!\n";
}
}
### Using the same adapter for both read 1 and read 2
else{
warn "\n >>> Now performing adapter trimming for the adapter sequence: '$adapter' from file $temp <<< \n";
# sleep (3);
$pid = open3 (\*WRITER, \*TRIM, \*ERROR,"$path_to_cutadapt $cores -e $error_rate -O $stringency -a $adapter $output_dir$temp") or die "Failed to launch Cutadapt: $!\n";
}
close WRITER or die $!; # not needed
open (QUAL,"$output_dir$temp") or die $!; # quality trimmed file
if ($filename =~ /\.gz$/){
open (IN,"$decompression_path -d -c $filename |") or die $!; # original, untrimmed file
}
else{
open (IN,$filename) or die $!; # original, untrimmed file
}
while (1){
# we can process the output from Cutadapt and the original input 1 by 1 to decide if the adapter has been removed or not
my $l1 = <TRIM>;
my $seq = <TRIM>; # adapter trimmed sequence
my $l3 = <TRIM>;
my $qual = <TRIM>;
$_ = <IN>; # irrelevant
my $original_seq = <IN>;
$_ = <IN>; # irrelevant
$_ = <IN>; # irrelevant
$_ = <QUAL>; # irrelevant
my $qual_trimmed_seq = <QUAL>;
$_ = <QUAL>; # irrelevant
my $qual_trimmed_qual = <QUAL>;
last unless (defined $qual and defined $qual_trimmed_qual); # could be empty strings
$count++;
if ($count%10000000 == 0){
warn "$count sequences processed\n";
}
chomp $seq;
chomp $qual;
chomp $qual_trimmed_seq;
chomp $original_seq;
my $quality_trimmed_seq_length = length $qual_trimmed_seq;
if (length $original_seq > length $qual_trimmed_seq){
++$quality_trimmed;
}
my $nd = 0;
### NON-DIRECTIONAL RRBS
if ($non_directional){
$nd = 1;
if (length$seq > 2){ # only doing something if the read is longer than 2bp