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bismark
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bismark
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#!/usr/bin/env perl
use strict;
use warnings;
use IO::Handle;
use Cwd;
$|++;
use Getopt::Long;
use FindBin qw($RealBin);
use lib "$RealBin/../lib";
## This program is Copyright (C) 2010-23, Felix Krueger (fkrueger@altoslabs.com)
## 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 version 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/>.
my $parent_dir = getcwd();
my $bismark_version = 'v0.24.2';
my $copyright_dates = "2010-23";
my $start_run = time();
my $command_line = join (" ",@ARGV);
### before processing the command line we will replace --solexa1.3-quals with --phred64-quals as the '.' in the option name will cause Getopt::Long to fail
foreach my $arg (@ARGV){
if ($arg eq '--solexa1.3-quals'){
$arg = '--phred64-quals';
}
}
my @filenames; # will be populated by processing the command line
my ($genome_folder,$CT_index_basename,$GA_index_basename,$path_to_bowtie,$path_to_hisat2,$path_to_minimap2,$sequence_file_format,$aligner_options,
$directional,$unmapped,$ambiguous,$phred64,$output_dir,$bowtie2,$hisat2,$sam_no_hd,$skip,$upto,$temp_dir,$non_bs_mm,$insertion_open,
$insertion_extend,$deletion_open,$deletion_extend,$gzip,$bam,$samtools_path,$pbat,$prefix,$old_flag,$basename,$score_min_intercept,
$score_min_slope,$bt2_large_index,$multicore,$rg_tag,$rg_id,$rg_sample,$ambig_bam,$cram,$cram_ref,$nucleotide_coverage,$dovetail,
$aligner_version,$slam,$icpc,$local,$strandID,$mm2,$maximum_length_cutoff) = process_command_line();
my @fhs; # stores alignment process names, bisulfite index location, bowtie filehandles and the number of times sequences produced an alignment
my %chromosomes; # stores the chromosome sequences of the mouse genome
my %SQ_order; # stores the order of sequences in the reference. This is to produce SAM/BAM files with a known order of chromosomes
my %counting; # counting various events
my $final_output_filename; # required for the nucleotide coverage report
my @pids; # storing the process IDs of child processes in parallel mode
my $seqID_contains_tabs;
my $verbose = 0;
if ($multicore > 1){
warn "Running Bismark Parallel version. Number of parallel instances to be spawned: $multicore\n\n";
}
sub multi_process_handling{
my $offset = 1;
my $process_id;
if ($multicore > 1){
until ($offset == $multicore){
# warn "multicore: $multicore\noffset: $offset\n";
my $fork = fork;
if (defined $fork){
if ($fork != 0){
$process_id = $fork;
push @pids, $process_id;
if ($offset < $multicore){
++$offset;
# warn "I am the parent process, child pid: $fork\nIncrementing offset counter to: $offset\n\n";
}
else{
# warn "Reached the number of maximum multicores. Proceeeding to processing...\n";
}
}
elsif ($fork == 0){
# warn "I am a child process, pid: $fork\nOffset counter is: $offset\nProceeding to processing...\n";
$process_id = $fork;
last;
}
}
else{
die "[FATAL ERROR]: Forking unsuccessful. This normally means that something is fundamentally not working with the fork command. Please run again without the --parallel option, or ask your system admin to look into this.\n";
}
}
# warn "\nThe Thread Identity\n===================\n";
if ($process_id){
# print "I am the parent process. My children are called:\n";
# print join ("\t",@pids),"\n";
# print "I am going to process the following line count: $offset\n\n";
}
elsif($process_id == 0){
# warn "I am a child process: Process ID: $process_id\n";
# warn "I am going to process the following line count: $offset\n\n";
}
else{
die "Process ID was: '$process_id'\n";
}
}
else{
warn "Single-core mode: setting pid to 1\n";
$process_id = 1;
}
return ($process_id,$offset);
}
sub subset_input_file_FastQ{
my ($filename,$process_id,$offset) = @_;
if ($filename =~ /gz$/){
open (OFFSET,"gunzip -c $filename |") or die "Couldn't read from file '$filename': $!\n";
}
else{
open (OFFSET,$filename) or die "Couldn't read from file '$filename': $!\n";
}
# warn "offset is $offset\n";
my $temp = $filename;
$temp .= ".temp.$offset";
$temp =~ s/^.*\///; # replacing everything upto and including the last /, i.e. removing file path information
if ($gzip){
$temp .= '.gz';
open (TEMPFQ,"| gzip -c - > ${temp_dir}${temp}") or die "Can't write to file ${temp_dir}${temp}: $!\n";
}
else{
open (TEMPFQ,'>',"${temp_dir}${temp}") or die "Failed to write output ${temp_dir}${temp}: $!\n";
}
my $line_count = 0;
my $seqs_processed = 0;
if (defined $upto){
# warn "Before we begin: -u was set to: -u $upto\n";
}
while (1){
my $l1 = <OFFSET>;
my $l2 = <OFFSET>;
my $l3 = <OFFSET>;
my $l4 = <OFFSET>;
last unless ($l4);
++$line_count;
# If user only want to process a subset of the input file
if (defined $upto){
if ($seqs_processed == $upto){
last;
}
}
if ( ($line_count - $offset)%$multicore == 0){
# warn "line count: $line_count\noffset: $offset\n";
# warn "Modulus: ",($line_count - $offset)%$multicore,"\n";
# warn "processing this line $line_count (processID: $process_id with \$offset $offset)\n";
print TEMPFQ "$l1$l2$l3$l4";
$seqs_processed++;
}
else{
# warn "skipping line $line_count for processID: $process_id with \$offset $offset)\n";
next;
}
}
close OFFSET; # or warn $!;
close TEMPFQ or warn "Failed to close file handle TEMPFQ: $!\n";
warn "Finished subdividing $filename for PID: $process_id and offset $offset (sequences written out: $seqs_processed)\n\n";
return ($temp); # returning the subset filename
}
sub subset_input_file_FastA{
my ($filename,$process_id,$offset) = @_;
if ($filename =~ /gz$/){
open (OFFSET,"gunzip -c $filename |") or die "Couldn't read from file '$filename': $!\n";
}
else{
open (OFFSET,$filename) or die "Couldn't read from file '$filename': $!\n";
}
# warn "offset is $offset\n";
my $temp = $filename;
$temp .= ".temp.$offset";
$temp =~ s/^.*\///; # replacing everything upto and including the last /, i.e. removing file path information
if ($gzip){
$temp .= '.gz';
open (TEMPFA,"| gzip -c - > ${temp_dir}${temp}") or die "Can't write to file ${temp_dir}${temp}: $!\n";
}
else{
open (TEMPFA,'>',"${temp_dir}${temp}") or die "Failed to write output ${temp_dir}${temp}: $!\n";
}
warn "Writing temporary infile to $temp\n";
my $line_count = 0;
my $seqs_processed = 0;
while (1){
my $l1 = <OFFSET>;
my $l2 = <OFFSET>;
last unless ($l2);
++$line_count;
# If user only want to process a subset of the input file
if (defined $upto){
if ($seqs_processed == $upto){
last;
}
}
if ( ($line_count - $offset)%$multicore == 0){
# warn "line count: $line_count\noffset: $offset\n";
# warn "Modulus: ",($line_count - $offset)%$multicore,"\n";
# warn "processing this line $line_count (processID: $process_id with \$offset $offset)\n";
print TEMPFA "$l1$l2";
$seqs_processed++;
}
else{
# warn "skipping line $line_count for processID: $process_id with \$offset $offset)\n";
next;
}
}
close OFFSET or warn $!;
close TEMPFA or warn "Failed to close file handle TEMPFQ: $!\n";
warn "Finished subdividing $filename for PID: $process_id and offset $offset (sequences processed: $seqs_processed)\n\n";
return ($temp); # returning the subset filename
}
####
####
foreach my $filename (@filenames){
my $original_filename = $filename;
my $original_filename_1;
my $original_filename_2;
chdir $parent_dir or die "Unable to move to initial working directory'$parent_dir' $!\n";
### resetting the counting hash and fhs
reset_counters_and_fhs($filename);
@pids = ();
$seqID_contains_tabs = 0;
### if 2 or more files are provided we can hold the genome in memory and don't need to read it in a second time
unless (%chromosomes){
my $cwd = getcwd(); # storing the path of the current working directory
warn "Current working directory is: $cwd\n\n";
read_genome_into_memory($cwd);
}
### As of version 0.14.0 we support multi-threading. In a first instance we accomplish this by
### splitting the input file(s) into several smaller subfiles and merging the results back at
### the end.
# get general settings (also for single-threaded use)
my ($pid,$offset) = multi_process_handling ();
my ($single_end,$paired_end);
### PAIRED-END ALIGNMENTS
if ($filename =~ ','){
$single_end = 0;
$paired_end = 1;
my ($C_to_T_infile_1,$G_to_A_infile_1); # to be made from mate1 file
$fhs[0]->{name} = 'CTread1GAread2CTgenome';
$fhs[1]->{name} = 'GAread1CTread2GAgenome';
$fhs[2]->{name} = 'GAread1CTread2CTgenome';
$fhs[3]->{name} = 'CTread1GAread2GAgenome';
warn "\nPaired-end alignments will be performed\n",'='x39,"\n\n";
my ($filename_1,$filename_2) = (split (/,/,$filename));
$original_filename_1 = $filename_1;
$original_filename_2 = $filename_2;
warn "The provided filenames for paired-end alignments are $filename_1 and $filename_2\n";
### subsetting the input file(s)
unless ($multicore == 1){ # not needed in single-core mode
# warn "My PID: $pid\nMy offset: $offset\n";
if ($sequence_file_format eq 'FASTA'){
my $temp_filename_1 = subset_input_file_FastA($filename_1,$pid,$offset);
warn "Using the subset file >${temp_dir}$temp_filename_1< as new in-file 1 (instead of >$filename_1<)\n";
$filename_1 = "${temp_dir}$temp_filename_1";
my $temp_filename_2 = subset_input_file_FastA($filename_2,$pid,$offset);
warn "Using the subset file >${temp_dir}$temp_filename_2< as new in-file 2 (instead of >$filename_2<)\n";
$filename_2 = "${temp_dir}$temp_filename_2";
}
else{ # FastQ format, default
my $temp_filename_1 = subset_input_file_FastQ($filename_1,$pid,$offset);
warn "Using the subset file >${temp_dir}$temp_filename_1< as new in-file 1 (instead of >$filename_1<)\n";
$filename_1 = "${temp_dir}$temp_filename_1";
my $temp_filename_2 = subset_input_file_FastQ($filename_2,$pid,$offset);
warn "Using the subset file >${temp_dir}$temp_filename_2< as new in-file 2 (instead of >$filename_2<)\n";
$filename_2 = "${temp_dir}$temp_filename_2";
}
}
### additional variables only for paired-end alignments
my ($C_to_T_infile_2,$G_to_A_infile_2); # to be made from mate2 file
my $read1_count; # to see if R1 and R2 have the same length
my $read2_count;
### FastA format
if ($sequence_file_format eq 'FASTA'){
warn "Input files are in FastA format\n";
if ($directional){
($C_to_T_infile_1,$read1_count) = biTransformFastAFiles_paired_end ($filename_1,1); # also passing the read number
($G_to_A_infile_2,$read2_count) = biTransformFastAFiles_paired_end ($filename_2,2);
$fhs[0]->{inputfile_1} = $C_to_T_infile_1;
$fhs[0]->{inputfile_2} = $G_to_A_infile_2;
$fhs[1]->{inputfile_1} = undef;
$fhs[1]->{inputfile_2} = undef;
$fhs[2]->{inputfile_1} = undef;
$fhs[2]->{inputfile_2} = undef;
$fhs[3]->{inputfile_1} = $C_to_T_infile_1;
$fhs[3]->{inputfile_2} = $G_to_A_infile_2;
}
elsif($pbat){ # PBAT-Seq
($G_to_A_infile_1,$read1_count) = biTransformFastAFiles_paired_end ($filename_1,1); # also passing the read number
($C_to_T_infile_2,$read2_count) = biTransformFastAFiles_paired_end ($filename_2,2);
$fhs[0]->{inputfile_1} = undef;
$fhs[0]->{inputfile_2} = undef;
$fhs[1]->{inputfile_1} = $G_to_A_infile_1;
$fhs[1]->{inputfile_2} = $C_to_T_infile_2;
$fhs[2]->{inputfile_1} = $G_to_A_infile_1;
$fhs[2]->{inputfile_2} = $C_to_T_infile_2;
$fhs[3]->{inputfile_1} = undef;
$fhs[3]->{inputfile_2} = undef;
}
else{
($C_to_T_infile_1,$G_to_A_infile_1,$read1_count) = biTransformFastAFiles_paired_end ($filename_1,1); # also passing the read number
($C_to_T_infile_2,$G_to_A_infile_2,$read2_count) = biTransformFastAFiles_paired_end ($filename_2,2);
$fhs[0]->{inputfile_1} = $C_to_T_infile_1;
$fhs[0]->{inputfile_2} = $G_to_A_infile_2;
$fhs[1]->{inputfile_1} = $G_to_A_infile_1;
$fhs[1]->{inputfile_2} = $C_to_T_infile_2;
$fhs[2]->{inputfile_1} = $G_to_A_infile_1;
$fhs[2]->{inputfile_2} = $C_to_T_infile_2;
$fhs[3]->{inputfile_1} = $C_to_T_infile_1;
$fhs[3]->{inputfile_2} = $G_to_A_infile_2;
}
unless ($read1_count eq $read2_count){
die "[FATAL ERROR]:\tNumber of bisulfite transformed reads are not equal between Read 1 (\#$read1_count) and Read 2 (\#$read2_count).\nPossible causes: file truncation, or as a result of specifying read pairs that do not belong to each other?! Please re-specify file names! Exiting...\n\n";
}
if ($bowtie2){
paired_end_align_fragments_to_bisulfite_genome_fastA_bowtie2 ($C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2);
}
else{ # HISAT2
paired_end_align_fragments_to_bisulfite_genome_fastA_hisat2 ($C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2);
}
}
### FastQ format
else{
warn "Input files are in FastQ format\n";
if ($directional){
if ($slam){
($C_to_T_infile_1,$read1_count) = biTransformFastQFiles_paired_end_slam ($filename_1,1); # also passing the read number
($G_to_A_infile_2,$read2_count) = biTransformFastQFiles_paired_end_slam ($filename_2,2);
}
else{
($C_to_T_infile_1,$read1_count) = biTransformFastQFiles_paired_end ($filename_1,1); # also passing the read number
($G_to_A_infile_2,$read2_count) = biTransformFastQFiles_paired_end ($filename_2,2);
}
$fhs[0]->{inputfile_1} = $C_to_T_infile_1;
$fhs[0]->{inputfile_2} = $G_to_A_infile_2;
$fhs[1]->{inputfile_1} = undef;
$fhs[1]->{inputfile_2} = undef;
$fhs[2]->{inputfile_1} = undef;
$fhs[2]->{inputfile_2} = undef;
$fhs[3]->{inputfile_1} = $C_to_T_infile_1;
$fhs[3]->{inputfile_2} = $G_to_A_infile_2;
}
elsif($pbat){ # PBAT-Seq
### At the moment we are only performing alignments only with uncompressed FastQ files
if ($slam){
($G_to_A_infile_1,$read1_count) = biTransformFastQFiles_paired_end_slam ($filename_1,1); # also passing the read number
($C_to_T_infile_2,$read2_count) = biTransformFastQFiles_paired_end_slam ($filename_2,2);
}
else{
($G_to_A_infile_1,$read1_count) = biTransformFastQFiles_paired_end ($filename_1,1); # also passing the read number
($C_to_T_infile_2,$read2_count) = biTransformFastQFiles_paired_end ($filename_2,2);
}
$fhs[0]->{inputfile_1} = undef;
$fhs[0]->{inputfile_2} = undef;
$fhs[1]->{inputfile_1} = $G_to_A_infile_1;
$fhs[1]->{inputfile_2} = $C_to_T_infile_2;
$fhs[2]->{inputfile_1} = $G_to_A_infile_1;
$fhs[2]->{inputfile_2} = $C_to_T_infile_2;
$fhs[3]->{inputfile_1} = undef;
$fhs[3]->{inputfile_2} = undef;
}
else{ # non-directional
if ($slam){
($C_to_T_infile_1,$G_to_A_infile_1,$read1_count) = biTransformFastQFiles_paired_end_slam ($filename_1,1); # also passing the read number
($C_to_T_infile_2,$G_to_A_infile_2,$read2_count) = biTransformFastQFiles_paired_end_slam ($filename_2,2);
}
else{
($C_to_T_infile_1,$G_to_A_infile_1,$read1_count) = biTransformFastQFiles_paired_end ($filename_1,1); # also passing the read number
($C_to_T_infile_2,$G_to_A_infile_2,$read2_count) = biTransformFastQFiles_paired_end ($filename_2,2);
}
$fhs[0]->{inputfile_1} = $C_to_T_infile_1;
$fhs[0]->{inputfile_2} = $G_to_A_infile_2;
$fhs[1]->{inputfile_1} = $G_to_A_infile_1;
$fhs[1]->{inputfile_2} = $C_to_T_infile_2;
$fhs[2]->{inputfile_1} = $G_to_A_infile_1;
$fhs[2]->{inputfile_2} = $C_to_T_infile_2;
$fhs[3]->{inputfile_1} = $C_to_T_infile_1;
$fhs[3]->{inputfile_2} = $G_to_A_infile_2;
}
unless ($read1_count eq $read2_count){
die "[FATAL ERROR]:\tNumber of bisulfite transformed reads are not equal between Read 1 (\#$read1_count) and Read 2 (\#$read2_count).\nPossible causes: file truncation, or as a result of specifying read pairs that do not belong to each other?! Please re-specify file names! Exiting...\n\n";
}
if ($bowtie2){
paired_end_align_fragments_to_bisulfite_genome_fastQ_bowtie2 ($C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2);
}
elsif ($mm2){
paired_end_align_fragments_to_bisulfite_genome_fastQ_minimap2 ($C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2);
}
else{ #
paired_end_align_fragments_to_bisulfite_genome_fastQ_hisat2 ($C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2);
}
}
start_methylation_call_procedure_paired_ends($filename_1,$filename_2,$C_to_T_infile_1,$G_to_A_infile_1,$C_to_T_infile_2,$G_to_A_infile_2,$pid);
}
### Else we are performing SINGLE-END ALIGNMENTS
else{
warn "\nSingle-end alignments will be performed\n",'='x39,"\n\n";
$single_end = 1;
$paired_end = 0;
### subsetting the input file(s)
unless ($multicore == 1){ # not needed in single-core mode
# warn "My PID: $pid\nMy offset: $offset\n";
if ($sequence_file_format eq 'FASTA'){
my $temp_filename = subset_input_file_FastA($filename,$pid,$offset);
warn "Using the subset file >${temp_dir}$temp_filename< as new in-file (instead of >$filename<)\n";
$filename = "${temp_dir}$temp_filename";
}
else{ # FastQ format, default
my $temp_filename = subset_input_file_FastQ($filename,$pid,$offset);
warn "Using the subset file >${temp_dir}$temp_filename< as new in-file (instead of >$filename<)\n";
$filename = "${temp_dir}$temp_filename";
}
}
### Initialising bisulfite conversion filenames
my ($C_to_T_infile,$G_to_A_infile);
### FastA format
if ($sequence_file_format eq 'FASTA'){
warn "Input file is in FastA format\n";
if ($directional){
($C_to_T_infile) = biTransformFastAFiles ($filename);
$fhs[0]->{inputfile} = $fhs[1]->{inputfile} = $C_to_T_infile;
}
else{
($C_to_T_infile,$G_to_A_infile) = biTransformFastAFiles ($filename);
$fhs[0]->{inputfile} = $fhs[1]->{inputfile} = $C_to_T_infile;
$fhs[2]->{inputfile} = $fhs[3]->{inputfile} = $G_to_A_infile;
}
### Creating 4 different bowtie filehandles and storing the first entry
if ($bowtie2){
single_end_align_fragments_to_bisulfite_genome_fastA_bowtie2 ($C_to_T_infile,$G_to_A_infile);
}
else{
single_end_align_fragments_to_bisulfite_genome_fastA_hisat2 ($C_to_T_infile,$G_to_A_infile);
}
}
## FastQ format
else{
warn "Input file is in FastQ format\n";
if ($directional){
if ($slam){
($C_to_T_infile) = biTransformFastQFiles_slam ($filename);
}
else{
($C_to_T_infile) = biTransformFastQFiles ($filename);
}
$fhs[0]->{inputfile} = $fhs[1]->{inputfile} = $C_to_T_infile;
}
elsif($pbat){
if ($slam){
($G_to_A_infile) = biTransformFastQFiles_slam ($filename);
}
else{
($G_to_A_infile) = biTransformFastQFiles ($filename);
}
$fhs[0]->{inputfile} = $fhs[1]->{inputfile} = $G_to_A_infile; # PBAT-Seq only uses the G to A converted files
}
else{
if ($slam){
($C_to_T_infile,$G_to_A_infile) = biTransformFastQFiles_slam ($filename);
}
else{
($C_to_T_infile,$G_to_A_infile) = biTransformFastQFiles ($filename);
}
$fhs[0]->{inputfile} = $fhs[1]->{inputfile} = $C_to_T_infile;
$fhs[2]->{inputfile} = $fhs[3]->{inputfile} = $G_to_A_infile;
}
### Creating up to 4 different filehandles and storing the first entry
if ($pbat){
if ($bowtie2){ # as of version 0.10.2 we also support PBAT alignments for Bowtie 2
single_end_align_fragments_to_bisulfite_genome_fastQ_bowtie2 (undef,$G_to_A_infile);
}
elsif($mm2){
single_end_align_fragments_to_bisulfite_genome_fastQ_minimap2 (undef,$G_to_A_infile);
}
else{ # HISAT2
single_end_align_fragments_to_bisulfite_genome_fastQ_hisat2 (undef,$G_to_A_infile);
}
}
elsif ($bowtie2){
single_end_align_fragments_to_bisulfite_genome_fastQ_bowtie2 ($C_to_T_infile,$G_to_A_infile);
}
elsif($mm2){
# warn "Now kicking off the single-end Minimap2 alignments\n\n"; sleep(3);
single_end_align_fragments_to_bisulfite_genome_fastQ_minimap2 ($C_to_T_infile,$G_to_A_infile);
}
else{ # HISAT2
single_end_align_fragments_to_bisulfite_genome_fastQ_hisat2 ($C_to_T_infile,$G_to_A_infile);
}
}
start_methylation_call_procedure_single_ends($filename,$C_to_T_infile,$G_to_A_infile,$pid);
}
### MERGING AND DELETING TEMP FILES // TIDYING UP AFTER A MULTICORE PROCESS
if ($pid){ # only performing this for the parent process
if ($multicore > 1){
warn "Now waiting for all child processes to complete\n";
### we need to ensure that we wait for all child processes to be finished before continuing
# warn "here are the child IDs: @pids\n";
# warn "Looping through the child process IDs:\n";
my $all_children_succeeded = 1;
foreach my $id (@pids){
# print "$id\t";
my $kid = waitpid ($id,0);
# print "Returned: $kid\nExit status: $?\n";
unless ($? == 0){
$all_children_succeeded = 0;
warn "\nChild process terminated with exit signal: '$?'\n\n";
}
}
if ($all_children_succeeded) {
print "All child process successfully finished.";
}
else {
die "\nTerminating. Not all child processes successfully finished.";
}
# regenerating names for temporary files
my @temp_input;
my @temp_output;
my @temp_reports;
my @temp_unmapped_1; # will store single end reads or R1 of paired-end
my @temp_unmapped_2;
my @temp_ambiguous_1; # will store single end reads or R1 of paired-end
my @temp_ambiguous_2;
my @temp_ambig_bam;
for (1..$offset){
# Temp Input Files
if ($single_end){
if ($gzip){
push @temp_input, "${original_filename}.temp.${_}.gz";
}
else{
push @temp_input, "${original_filename}.temp.${_}";
}
}
elsif($paired_end){
if ($gzip){
push @temp_input, "${original_filename_1}.temp.${_}.gz";
push @temp_input, "${original_filename_2}.temp.${_}.gz";
}
else{
push @temp_input, "${original_filename_1}.temp.${_}";
push @temp_input, "${original_filename_2}.temp.${_}";
}
}
# if files had a prefix we need to specify it
my $add_prefix;
if (defined $prefix){
$add_prefix = "${prefix}.";
}
else{
$add_prefix = '';
}
if ($single_end){
# Temp Output Files
my $pathless_filename = ${original_filename}; # 10 Jan 2017
$pathless_filename =~ s/.*\///; # deleting path information
if ($bowtie2){
if ($gzip){
push @temp_output, "${output_dir}${add_prefix}${pathless_filename}.temp.${_}.gz_bismark_bt2.bam";
push @temp_reports, "${output_dir}${add_prefix}${pathless_filename}.temp.${_}.gz_bismark_bt2_SE_report.txt";
push @temp_ambig_bam, "${output_dir}${add_prefix}${pathless_filename}.temp.${_}.gz_bismark_bt2.ambig.bam"; # only for Bowtie 2
}
else{
push @temp_output, "${output_dir}${add_prefix}${pathless_filename}.temp.${_}_bismark_bt2.bam";
push @temp_reports, "${output_dir}${add_prefix}${pathless_filename}.temp.${_}_bismark_bt2_SE_report.txt";
push @temp_ambig_bam, "${output_dir}${add_prefix}${pathless_filename}.temp.${_}_bismark_bt2.ambig.bam"; # only for Bowtie 2
}
}
elsif($mm2){
if ($gzip){
push @temp_output, "${output_dir}${add_prefix}${pathless_filename}.temp.${_}.gz_bismark_mm22.bam";
push @temp_reports, "${output_dir}${add_prefix}${pathless_filename}.temp.${_}.gz_bismark_mm2_SE_report.txt";
}
else{
push @temp_output, "${output_dir}${add_prefix}${pathless_filename}.temp.${_}_bismark_mm2.bam";
push @temp_reports, "${output_dir}${add_prefix}${pathless_filename}.temp.${_}_bismark_mm2_SE_report.txt";
}
}
else{ # HISAT2
if ($gzip){
push @temp_output, "${output_dir}${add_prefix}${pathless_filename}.temp.${_}.gz_bismark_hisat2.bam";
push @temp_reports, "${output_dir}${add_prefix}${pathless_filename}.temp.${_}.gz_bismark_hisat2_SE_report.txt";
}
else{
push @temp_output, "${output_dir}${add_prefix}${pathless_filename}.temp.${_}_bismark_hisat2.bam";
push @temp_reports, "${output_dir}${add_prefix}${pathless_filename}.temp.${_}_bismark_hisat2_SE_report.txt";
}
}
if ($unmapped){
if ($gzip){
push @temp_unmapped_1, "${output_dir}${add_prefix}${pathless_filename}.temp.${_}.gz_unmapped_reads.fq";
}
else{
push @temp_unmapped_1, "${output_dir}${add_prefix}${pathless_filename}.temp.${_}_unmapped_reads.fq";
}
}
if ($ambiguous){
if ($gzip){
push @temp_ambiguous_1, "${output_dir}${add_prefix}${pathless_filename}.temp.${_}.gz_ambiguous_reads.fq";
}
else{
push @temp_ambiguous_1, "${output_dir}${add_prefix}${pathless_filename}.temp.${_}_ambiguous_reads.fq";
}
}
}
elsif($paired_end){
# Temp Output Files
my $pathless_filename_1 = ${original_filename_1}; # 10 Jan 2017
my $pathless_filename_2 = ${original_filename_2};
$pathless_filename_1 =~ s/.*\///; # deleting path information
$pathless_filename_2 =~ s/.*\///; # deleting path information
if ($bowtie2){
if ($gzip){
push @temp_output, "${output_dir}${add_prefix}${pathless_filename_1}.temp.${_}.gz_bismark_bt2_pe.bam";
push @temp_reports, "${output_dir}${add_prefix}${pathless_filename_1}.temp.${_}.gz_bismark_bt2_PE_report.txt";
push @temp_ambig_bam, "${output_dir}${add_prefix}${pathless_filename_1}.temp.${_}.gz_bismark_bt2_pe.ambig.bam"; # only for Bowtie 2
}
else{
push @temp_output, "${output_dir}${add_prefix}${pathless_filename_1}.temp.${_}_bismark_bt2_pe.bam";
push @temp_reports, "${output_dir}${add_prefix}${pathless_filename_1}.temp.${_}_bismark_bt2_PE_report.txt";
push @temp_ambig_bam, "${output_dir}${add_prefix}${pathless_filename_1}.temp.${_}_bismark_bt2_pe.ambig.bam"; # only for Bowtie 2
}
}
elsif($mm2){
if ($gzip){
push @temp_output, "${output_dir}${add_prefix}${pathless_filename_1}.temp.${_}.gz_bismark_mm2_pe.bam";
push @temp_reports, "${output_dir}${add_prefix}${pathless_filename_1}.temp.${_}.gz_bismark_mm2_PE_report.txt";
}
else{
push @temp_output, "${output_dir}${add_prefix}${pathless_filename_1}.temp.${_}_bismark_mm2_pe.bam";
push @temp_reports, "${output_dir}${add_prefix}${pathless_filename_1}.temp.${_}_bismark_mm2_PE_report.txt";
}
}
else{
if ($gzip){
push @temp_output, "${output_dir}${add_prefix}${pathless_filename_1}.temp.${_}.gz_bismark_hisat2_pe.bam";
push @temp_reports, "${output_dir}${add_prefix}${pathless_filename_1}.temp.${_}.gz_bismark_hisat2_PE_report.txt";
}
else{
push @temp_output, "${output_dir}${add_prefix}${pathless_filename_1}.temp.${_}_bismark_hisat2_pe.bam";
push @temp_reports, "${output_dir}${add_prefix}${pathless_filename_1}.temp.${_}_bismark_hisat2_PE_report.txt";
}
}
if ($unmapped){
if ($gzip){
push @temp_unmapped_1, "${output_dir}${add_prefix}${pathless_filename_1}.temp.${_}.gz_unmapped_reads_1.fq";
push @temp_unmapped_2, "${output_dir}${add_prefix}${pathless_filename_2}.temp.${_}.gz_unmapped_reads_2.fq";
}
else{
push @temp_unmapped_1, "${output_dir}${add_prefix}${pathless_filename_1}.temp.${_}_unmapped_reads_1.fq";
push @temp_unmapped_2, "${output_dir}${add_prefix}${pathless_filename_2}.temp.${_}_unmapped_reads_2.fq";
}
}
if ($ambiguous){
if ($gzip){
push @temp_ambiguous_1, "${output_dir}${add_prefix}${pathless_filename_1}.temp.${_}.gz_ambiguous_reads_1.fq";
push @temp_ambiguous_2, "${output_dir}${add_prefix}${pathless_filename_2}.temp.${_}.gz_ambiguous_reads_2.fq";
}
else{
push @temp_ambiguous_1, "${output_dir}${add_prefix}${pathless_filename_1}.temp.${_}_ambiguous_reads_1.fq";
push @temp_ambiguous_2, "${output_dir}${add_prefix}${pathless_filename_2}.temp.${_}_ambiguous_reads_2.fq";
}
}
}
}
warn "\n\nRight, cleaning up now...\n\n";
# deleting temp files;
warn "Deleting temporary sequence files...\n";
foreach my $temp (@temp_input){
#print "$temp\t";
$temp =~ s/.*\///; # deleting path information
print "${temp_dir}${temp}\t";
unlink "${temp_dir}${temp}" or warn "Failed to delete temporary FastQ file ${temp_dir}$temp: $!\n";
}
print "\n\n";
# merging temp BAM files
if ($single_end){
merge_individual_BAM_files(\@temp_output,$original_filename,$single_end);
}
else{
merge_individual_BAM_files(\@temp_output,$original_filename_1,$single_end);
}
# deleting temp BAM files
warn "Deleting temporary BAM files...\n";
foreach my $temp (@temp_output){
# print "$temp\t";
$temp =~ s/.*\///; # deleting path information
print "${output_dir}${temp}\t";
unlink "${output_dir}${temp}" or warn "Failed to delete temporary BAM file ${output_dir}${temp}: $!\n";
}
print "\n\n";
### AMBIGUOUS BAM files
if ($ambig_bam){
# merging temp AMBIG BAM files
if ($single_end){
merge_individual_ambig_BAM_files(\@temp_ambig_bam,$original_filename,$single_end);
}
else{
merge_individual_ambig_BAM_files(\@temp_ambig_bam,$original_filename_1,$single_end);
}
# deleting temp BAM files
warn "Deleting temporary ambiguous BAM files...\n";
foreach my $temp (@temp_ambig_bam){
# print "$temp\t";
$temp =~ s/.*\///; # deleting path information
print "${output_dir}${temp}\t";
unlink "${output_dir}${temp}" or warn "Failed to delete temporary ambiguous BAM file ${output_dir}${temp}: $!\n";
}
print "\n\n";
}
if ($unmapped){
if ($single_end){
merge_individual_unmapped_files(\@temp_unmapped_1,$original_filename,$single_end);
}
else{
merge_individual_unmapped_files(\@temp_unmapped_1,$original_filename_1,$single_end,'_1');
merge_individual_unmapped_files(\@temp_unmapped_2,$original_filename_2,$single_end,'_2');
}
# deleting temp unmapped files
warn "Deleting temporary unmapped files...\n";
foreach my $temp (@temp_unmapped_1){
print "$temp\t";
unlink "${output_dir}${temp}" or warn "Failed to delete temporary unmapped FastQ file ${output_dir}$temp: $!\n";
}
if ($paired_end){
foreach my $temp (@temp_unmapped_2){
print "$temp\t";
unlink "${output_dir}${temp}" or warn "Failed to delete temporary unmapped FastQ file ${output_dir}$temp: $!\n";
}
}
print "\n\n";
}
if ($ambiguous){
if ($single_end){
merge_individual_ambiguous_files(\@temp_ambiguous_1,$original_filename,$single_end);
}
else{
merge_individual_ambiguous_files(\@temp_ambiguous_1,$original_filename_1,$single_end,'_1');
merge_individual_ambiguous_files(\@temp_ambiguous_2,$original_filename_2,$single_end,'_2');
}
# deleting temp ambiguous files
warn "Deleting temporary ambiguous files...\n";
foreach my $temp (@temp_ambiguous_1){
print "$temp\t";
unlink "${output_dir}${temp}" or warn "Failed to delete temporary ambiguous FastQ file ${output_dir}$temp: $!\n";
}
if ($paired_end){
foreach my $temp (@temp_ambiguous_2){
print "$temp\t";
unlink "${output_dir}${temp}" or warn "Failed to delete temporary ambiguous FastQ file ${output_dir}$temp: $!\n";
}
}
print "\n\n";
}
# resetting the counters once more so we can add all data from all temporary reports
reset_counters_and_fhs($original_filename);
### Merging the Bismark mapping report files
if ($single_end){
merge_individual_mapping_reports(\@temp_reports,$original_filename,$single_end);
print_final_analysis_report_single_end('mock_file1','mock_file_2','mock_pid','mergeThis');
}
else{
merge_individual_mapping_reports(\@temp_reports,$original_filename_1,$single_end,$original_filename_2);
print_final_analysis_report_paired_ends('mock_file1','mock_file_2','mock_file3','mock_file_4','mock_pid','mergeThis');
}
# deleting temp report files
warn "Deleting temporary report files...\n";
foreach my $temp (@temp_reports){
print "$temp\t";
unlink "${output_dir}${temp}" or warn "Failed to delete temporary report file $output_dir$temp: $!\n";
}
print "\n\n";
}
}
if ($pid){ # only for the Parent
### Produce Run Time
my $end_run = time();
my $run_time = $end_run - $start_run;
my $days = int($run_time/(24*60*60));
my $hours = ($run_time/(60*60))%24;
my $mins = ($run_time/60)%60;
my $secs = $run_time%60;
warn "Bismark completed in ${days}d ${hours}h ${mins}m ${secs}s\n";
print REPORT "Bismark completed in ${days}d ${hours}h ${mins}m ${secs}s\n";
warn "\n====================\nBismark run complete\n====================\n\n";
if ($nucleotide_coverage){
warn "Now calculating observed and expected nucleotide coverage statistics... \n\n";
if ($final_output_filename =~ /(bam|cram)|/){
my @args;
push @args, "--genome $genome_folder";
push @args, "--dir '$output_dir'";
push @args, "--samtools_path $samtools_path";
push @args, $final_output_filename;
print "@args","\n"; sleep(3);
system ("$RealBin/bam2nuc @args");
warn "Finished bam2nuc calculation ...\n\n";
}
else{
warn "Nucleotide coverage statistics are currently only available for BAM or CRAM files\n\n";
}
}
# exit 0; # will terminate after a single supplied file....
}
else{
# If multiple files were supplied as the command line, like so:
# -1 R1.fastq,simulated_1.fastq,ZZZ_R1.fastq -2 R2.fastq,simulated_2.fastq,ZZZ_R2.fastq --multicore 4
# we need to exit from the child processes if we don't want a steady increase of new Bismark instances! Fixed 30 10 2017
# warn "Terminating Child process\n\n";
exit 0;
}
}
sub merge_individual_mapping_reports{
my ($temp_reports,$original_filename_1,$single_end,$original_filename_2) = @_;
my $report_file = $original_filename_1;
$report_file =~ s/.*\///; # removing path information
$report_file =~ s/(\.fastq\.gz|\.fq\.gz|\.fastq|\.fq)$//; # attempting to remove fastq.gz etc to make filename a little shorter
if ($prefix){
$report_file = "${prefix}.${report_file}";
}
if ($basename){ # Output file basename is set using the -B argument
$report_file = ${basename};
}
if ($single_end){
if ($bowtie2){
$report_file .= '_bismark_bt2_SE_report.txt';
}
elsif($mm2){
$report_file .= '_bismark_mm2_SE_report.txt';
}
else{
$report_file .= '_bismark_hisat2_SE_report.txt';
}
}
else{
if ($bowtie2){
$report_file .= '_bismark_bt2_PE_report.txt';
}
elsif($mm2){
$report_file .= '_bismark_mm2_PE_report.txt';
}
else{
$report_file .= '_bismark_hisat2_PE_report.txt';
}
}
warn "Writing report to ${output_dir}${report_file}\n";
open (REPORT,'>',"$output_dir$report_file") or die "Failed to write to ${output_dir}${report_file}: $!\n";
foreach my $temp(@$temp_reports){
$temp =~ s/.*\///; # removing path information
}
warn "Now merging temporary reports @$temp_reports into >>> ${output_dir}${report_file} <<<\n";
if ($single_end){
print REPORT "Bismark report for: $original_filename_1 (version: $bismark_version)\n";
}
else{ # paired-end
print REPORT "Bismark report for: $original_filename_1 and $original_filename_2 (version: $bismark_version)\n";
}
my $first = 0;
foreach my $temp(@$temp_reports){
# $temp =~ s/.*\///; # removing path information
warn "Merging from file >> $temp <<\n";
open (IN,"${output_dir}${temp}") or die "Failed to read from temporary mapping report '${output_dir}${temp}'\n";
### this is printing the first couple of lines
while (<IN>){
chomp;