#!/usr/bin/perl use warnings; use strict; use FindBin qw($RealBin); use Getopt::Long; ##################################################################################### # Copyright Copyright 2010-15 Simon Andrews # # # # This file is part of FastQC. # # # # FastQC is free software; you can redistribute it and/or modify # # it under the terms of the GNU General Public License as published by # # the Free Software Foundation; either version 3 of the License, or # # (at your option) any later version. # # # # FastQC is distributed in the hope that it will be useful, # # but WITHOUT ANY WARRANTY; without even the implied warranty of # # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # # GNU General Public License for more details. # # # # You should have received a copy of the GNU General Public License # # along with FastQC; if not, write to the Free Software # # Foundation, Inc., 51 Franklin St, Fifth Floor, Boston, MA 02110-1301 USA # ##################################################################################### # Check to see if they've mistakenly downloaded the source distribution # since several people have made this mistake if (-e "$RealBin/uk/ac/babraham/FastQC/FastQCApplication.java") { die "This is the source distribution of FastQC. You need to get the compiled version if you want to run the program\n"; } my $delimiter = ':'; if ($^O =~ /Win/) { $delimiter = ';'; } if ($ENV{CLASSPATH}) { $ENV{CLASSPATH} .= "$delimiter$RealBin$delimiter$RealBin/sam-1.103.jar$delimiter$RealBin/jbzip2-0.9.jar$delimiter$RealBin/cisd-jhdf5.jar"; } else { $ENV{CLASSPATH} = "$RealBin$delimiter$RealBin/sam-1.103.jar$delimiter$RealBin/jbzip2-0.9.jar$delimiter$RealBin/cisd-jhdf5.jar"; } my @java_args; my @files; # We now need to scan the command line for switches which we're going # to pass on to the main java program. my $version; my $help; my $outdir; my $unzip; my $format; my $contaminant; my $adapter; my $limits; my $threads; my $quiet; my $nogroup; my $expgroup; my $casava; my $nano; my $nofilter; my $kmer_size; my $temp_directory; my $java_bin = 'java'; my $result = GetOptions('version' => \$version, 'help' => \$help, 'quiet' => \$quiet, 'nogroup' => \$nogroup, 'expgroup' => \$expgroup, 'outdir=s' => \$outdir, 'extract!' => \$unzip, 'format=s' => \$format, 'threads=i' => \$threads, 'kmers=i' => \$kmer_size, 'casava' => \$casava, 'nano' => \$nano, 'nofilter' => \$nofilter, 'contaminants=s' => \$contaminant, 'adapters=s' => \$adapter, 'limits=s' => \$limits, 'dir=s' => \$temp_directory, 'java=s' => \$java_bin, ); # Check the simple stuff first if ($help) { # Just print the help and exit print while(); exit; } if ($version) { push @java_args ,"-Dfastqc.show_version=true"; } # Now parse any additional options if ($outdir) { unless(-e $outdir and -d $outdir) { die "Specified output directory '$outdir' does not exist\n"; } push @java_args ,"-Dfastqc.output_dir=$outdir"; } if ($contaminant) { unless (-e $contaminant and -r $contaminant) { die "Contaminant file '$contaminant' did not exist, or could not be read\n"; } push @java_args ,"-Dfastqc.contaminant_file=$contaminant"; } if ($adapter) { unless (-e $adapter and -r $adapter) { die "Adapters file '$adapter' did not exist, or could not be read\n"; } push @java_args ,"-Dfastqc.adapter_file=$adapter"; } if ($limits) { unless (-e $limits and -r $limits) { die "Limits file '$limits' did not exist, or could not be read\n"; } push @java_args ,"-Dfastqc.limits_file=$limits"; } if ($temp_directory) { unless (-e $temp_directory and -d $temp_directory and -w $temp_directory) { die "Temp directory '$temp_directory' doesn't exist, or can't be written to\n"; } push @java_args, "-Djava.io.tmpdir=$temp_directory"; } if ($threads) { if ($threads < 1) { die "Number of threads must be a positive integer"; } push @java_args ,"-Dfastqc.threads=$threads"; my $memory = 250 * $threads; unshift @java_args,"-Xmx${memory}m"; } else { unshift @java_args,'-Xmx250m'; } if ($kmer_size) { unless ($kmer_size =~ /^\d+$/) { die "Kmer size '$kmer_size' was not a number"; } if ($kmer_size < 2 or $kmer_size > 10) { die "Kmer size must be in the range 2-10"; } push @java_args,"-Dfastqc.kmer_size=$kmer_size"; } if ($quiet) { push @java_args ,"-Dfastqc.quiet=true"; } if ($casava) { push @java_args ,"-Dfastqc.casava=true"; } if ($nano) { push @java_args ,"-Dfastqc.nano=true"; } if ($nofilter) { push @java_args ,"-Dfastqc.nofilter=true"; } if ($nogroup) { push @java_args ,"-Dfastqc.nogroup=true"; } if ($expgroup) { if ($nogroup) { die "You can't specify both --expgroup and --nogroup in the same run\n"; } push @java_args ,"-Dfastqc.expgroup=true"; } if (defined $unzip) { if ($unzip) { $unzip = 'true'; } else { $unzip = 'false'; } push @java_args,"-Dfastqc.unzip=$unzip"; } if ($format) { unless ($format eq 'bam' || $format eq 'sam' || $format eq 'fastq' || $format eq 'sam_mapped' || $format eq 'bam_mapped') { die "Unrecognised sequence format '$format', acceptable formats are bam,sam,bam_mapped,sam_mapped and fastq\n"; } push @java_args,"-Dfastqc.sequence_format=$format"; } if ($java_bin ne 'java') { # $java_bin =~ s/\\/\//g; unless (-e $java_bin) { die "Couldn't find java interpreter at '$java_bin'"; } # if ($java_bin =~ / /) { # $java_bin = "\"$java_bin\""; # } } # We've found that on systems with large numbers of CPUs, and especially on systems # where IO contention can occur, that the program can consume large amounts of CPU # during parallel garbage collection. Since interactive performance isn't really # important when running non-interactively we'll limit the GC to using only a single # thread if running with a list of filenames if (@ARGV) { push @java_args,"-XX:ParallelGCThreads=1"; } foreach (@ARGV) { if (/^\-D/) { push @java_args,$_; } else { push @files,$_; } } # This is set internally as well, but on some JREs it doesn't # pick up the internally set value properly, so we'll set it # outside as well which should work. if (@files or $version or $help) { push @java_args, "-Djava.awt.headless=true"; } #warn "Running 'exec $java_bin,@java_args, \"uk.ac.babraham.FastQC.FastQCApplication\", @files, CLASSPATH is $ENV{CLASSPATH}"; if ($java_bin ne 'java') { system $java_bin,@java_args, "uk.ac.babraham.FastQC.FastQCApplication", @files; } else { exec $java_bin,@java_args, "uk.ac.babraham.FastQC.FastQCApplication", @files; } __DATA__ FastQC - A high throughput sequence QC analysis tool SYNOPSIS fastqc seqfile1 seqfile2 .. seqfileN fastqc [-o output dir] [--(no)extract] [-f fastq|bam|sam] [-c contaminant file] seqfile1 .. seqfileN DESCRIPTION FastQC reads a set of sequence files and produces from each one a quality control report consisting of a number of different modules, each one of which will help to identify a different potential type of problem in your data. If no files to process are specified on the command line then the program will start as an interactive graphical application. If files are provided on the command line then the program will run with no user interaction required. In this mode it is suitable for inclusion into a standardised analysis pipeline. The options for the program as as follows: -h --help Print this help file and exit -v --version Print the version of the program and exit -o --outdir Create all output files in the specified output directory. Please note that this directory must exist as the program will not create it. If this option is not set then the output file for each sequence file is created in the same directory as the sequence file which was processed. --casava Files come from raw casava output. Files in the same sample group (differing only by the group number) will be analysed as a set rather than individually. Sequences with the filter flag set in the header will be excluded from the analysis. Files must have the same names given to them by casava (including being gzipped and ending with .gz) otherwise they won't be grouped together correctly. --nano Files come from naopore sequences and are in fast5 format. In this mode you can pass in directories to process and the program will take in all fast5 files within those directories and produce a single output file from the sequences found in all files. --nofilter If running with --casava then don't remove read flagged by casava as poor quality when performing the QC analysis. --extract If set then the zipped output file will be uncompressed in the same directory after it has been created. By default this option will be set if fastqc is run in non-interactive mode. -j --java Provides the full path to the java binary you want to use to launch fastqc. If not supplied then java is assumed to be in your path. --noextract Do not uncompress the output file after creating it. You should set this option if you do not wish to uncompress the output when running in non-interactive mode. --nogroup Disable grouping of bases for reads >50bp. All reports will show data for every base in the read. WARNING: Using this option will cause fastqc to crash and burn if you use it on really long reads, and your plots may end up a ridiculous size. You have been warned! -f --format Bypasses the normal sequence file format detection and forces the program to use the specified format. Valid formats are bam,sam,bam_mapped,sam_mapped and fastq -t --threads Specifies the number of files which can be processed simultaneously. Each thread will be allocated 250MB of memory so you shouldn't run more threads than your available memory will cope with, and not more than 6 threads on a 32 bit machine -c Specifies a non-default file which contains the list of --contaminants contaminants to screen overrepresented sequences against. The file must contain sets of named contaminants in the form name[tab]sequence. Lines prefixed with a hash will be ignored. -a Specifies a non-default file which contains the list of --adapters adapter sequences which will be explicity searched against the library. The file must contain sets of named adapters in the form name[tab]sequence. Lines prefixed with a hash will be ignored. -l Specifies a non-default file which contains a set of criteria --limits which will be used to determine the warn/error limits for the various modules. This file can also be used to selectively remove some modules from the output all together. The format needs to mirror the default limits.txt file found in the Configuration folder. -k --kmers Specifies the length of Kmer to look for in the Kmer content module. Specified Kmer length must be between 2 and 10. Default length is 7 if not specified. -q --quiet Supress all progress messages on stdout and only report errors. -d --dir Selects a directory to be used for temporary files written when generating report images. Defaults to system temp directory if not specified. BUGS Any bugs in fastqc should be reported either to simon.andrews@babraham.ac.uk or in www.bioinformatics.babraham.ac.uk/bugzilla/