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1 #!/usr/bin/perl
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2 use strict;
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3 use warnings;
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4 use Getopt::Long;
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5 use Parallel::ForkManager;
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6 use File::Basename;
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7 use File::Copy::Recursive qw( dircopy );
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8 use POSIX;
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9 use FindBin;
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10 use lib $FindBin::Bin;
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11 use resize qw ( size_distribution );
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12 use subgroups qw (subgroups );
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13 use ppp qw ( ping_pong_partners );
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14 use Rcall qw (pie_chart bg_to_png );
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23
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15 use align qw ( to_build get_unique sam_count sam_count_mis sam_sorted_bam rpms_rpkm rpms_rpkm_te BWA_call get_fastq_seq extract_sam sam_to_bam_bg );
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1
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16 use html qw ( main_page details_pages menu_page ppp_page );
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17 use File::Copy;
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18
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19 my ( @fastq, @fastq_n, $dir, $min, $max, $mis, $misTE, $help, $Pcheck, $mapnumf, $html_out);
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20 my ( $ref, $tRNAs, $rRNAs, $snRNAs, $miRNAs, $transcripts, $TE );
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21 my ( $si_min, $si_max, $pi_min, $pi_max );
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22 my ( $build_index, $build_tRNAs, $build_rRNAs, $build_snRNAs, $build_miRNAs, $build_transcripts, $build_TE );
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23 my $max_procs = 8;
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24
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25 ( $build_index, $build_tRNAs, $build_rRNAs, $build_snRNAs, $build_miRNAs, $build_transcripts, $build_TE ) = (0,0,0,0,0,0,0);
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26 ( $min, $max, $mis, $misTE, $si_min, $si_max, $pi_min, $pi_max, $dir ) = ( 18, 29, 0, 3, 21, 21, 23, 29 );
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27 $Pcheck ='true';
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28
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29 GetOptions (
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30 "fastq=s" => \@fastq,
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31 "fastq_n=s" => \@fastq_n,
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32 "dir=s" => \$dir,
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33 "min:i" => \$min,
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34 "max:i" => \$max,
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35 "si_min:i" => \$si_min,
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36 "si_max:i" => \$si_max,
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37 "pi_min:i" => \$pi_min,
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38 "pi_max:i" => \$pi_max,
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39 "mis:i" => \$mis,
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40 "misTE:i" => \$misTE,
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41 "html:s" => \$html_out,
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42 "PPPon:s" => \$Pcheck,
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43 "help" => \$help,
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44 "ref:s" => \$ref,
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45 "tRNAs:s" => \$tRNAs,
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46 "rRNAs:s" => \$rRNAs,
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47 "snRNAs:s" => \$snRNAs,
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48 "miRNAs:s" => \$miRNAs,
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49 "transcripts:s" => \$transcripts,
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50 "TE:s" => \$TE,
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51 "build_index" => \$build_index,
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52 "build_tRNAs" => \$build_tRNAs,
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53 "build_snRNAs" => \$build_snRNAs,
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54 "build_miRNAs" => \$build_miRNAs,
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55 "build_transcripts" => \$build_transcripts,
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56 "build_rRNAs" => \$build_rRNAs,
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57 "build_TE" => \$build_TE
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58 );
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59
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60 my $fq_collection = 'fastq_dir/';
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61 mkdir $dir; mkdir $fq_collection;
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62 $dir = $dir.'/' unless $dir =~ /\/$/;
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63 mkdir $dir.'/css';mkdir $dir.'/js';
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64 dircopy( $FindBin::Bin.'/css', $dir.'/css' );
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65 dircopy( $FindBin::Bin.'/js', $dir.'/js' );
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66
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67 my $file = $dir.'report.txt';
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68 open my $report, '>', $file or die "Cannot open $file $!\n";
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69
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70 my @toBuild = ( [$build_index, \$ref], [$build_tRNAs, \$tRNAs], [$build_rRNAs, \$rRNAs], [$build_snRNAs, \$snRNAs], [$build_miRNAs, \$miRNAs], [$build_transcripts, \$transcripts], [$build_TE, \$TE] );
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71 to_build ( \@toBuild, $report, $dir );
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72
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73 my $proc_child = ceil($max_procs / scalar(@fastq));
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74 my $proc_grand_child = ceil($proc_child/4);
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75 my $pm = Parallel::ForkManager->new($max_procs);
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76 my $pm2 = Parallel::ForkManager->new($proc_grand_child);
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77
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78 $pm->run_on_finish( sub {
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79 my ($pid, $exit_code, $ident) = @_;
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80 print $report "Fastq fork $ident just finished ".
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81 "with PID $pid and exit code: $exit_code\n";
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82 die "Something went wrong!\n" if $exit_code != 0;
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83 });
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84 $pm->run_on_start( sub {
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85 my ($pid,$ident)=@_;
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86 print $report "Fastq fork : $ident started, pid: $pid\n";
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87 });
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88 $pm2->run_on_finish( sub {
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89 my ($pid, $exit_code, $ident) = @_;
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90 print $report "** Subgroup fork $ident just finished ".
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91 "with PID $pid and exit code: $exit_code\n";
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92 die "Something went wrong!\n" if $exit_code != 0;
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93 });
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94 $pm2->run_on_start( sub {
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95 my ($pid,$ident)=@_;
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96 print $report "** Subgroup fork $ident started, pid: $pid\n";
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97 });
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98
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99
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100 foreach my $child ( 0 .. $#fastq )
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101 {
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102 my @suffix = ('.fastq', '.fastq.gz,', '.fq', '.fq.gz', 'ref', '.dat', '.fa','.fas','.fasta', '.txt');
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103 my ( $name, $path, $suffix ) = fileparse( $fastq[$child], @suffix );
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104 my ( $ref_name, $ref_path, $ref_suffix ) = fileparse( $ref, @suffix );
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105 my ( $TE_name, $TE_path, $TE_suffix ) = fileparse( $TE, @suffix );
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106 my ( $ex_name, $ex_path, $ex_suffix ) = fileparse( $transcripts, @suffix );
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107
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108 $pm->start($fastq[$child]) and next;
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109
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110 my $dir_fq = $dir.$fastq_n[$child].'/';
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111 mkdir $dir_fq;
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112
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113 my $gen_dir = $dir_fq.'genome/';
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114 mkdir $gen_dir;
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115
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116 my $size_dir = $dir_fq.'size/';
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117 mkdir $size_dir;
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118
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119 my $fastq_resized = $dir_fq.$name.'_'.$min.'-'.$max.'.fastq';
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120 size_distribution ( $fastq[$child], $fastq_resized, $size_dir, $min, $max );
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121
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122 my $sam_genome = $gen_dir.$fastq_n[$child].'_'.$min.'-'.$max.'.sam';
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123 my $sam_genome_unique = $gen_dir.$fastq_n[$child].'_'.$min.'-'.$max.'_unique.sam';
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124 my $fastq_prefix = $gen_dir.$fastq_n[$child].'_'.$min.'-'.$max;
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125
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126 BWA_call ( $ref, $fastq_resized, $sam_genome, $mis, $proc_child, $report );
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127 my ( $fai_ref_hashP, $ma, $ma_uni ) = get_unique ( $sam_genome, $sam_genome_unique, $gen_dir, $fq_collection.$fastq_n[$child], 1, $report );
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128
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129 die "No Reads mapped on the genome reference!\n" if $ma == 0;
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130 my $scale = 1000000 / $ma;
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131 sam_to_bam_bg ( $sam_genome_unique, $scale, $proc_child );
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132 sam_to_bam_bg ( $sam_genome, $scale, $proc_child );
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133
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134 my $Gviz_dir = $gen_dir.'Gviz/';
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135 my $fai_file = $gen_dir.'fai';
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136 mkdir $Gviz_dir;
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137 my $Gviz_dir_rand = $Gviz_dir.'rand/';
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138 mkdir $Gviz_dir_rand;
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139 my $Gviz_dir_uni = $Gviz_dir.'unique/';
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140 mkdir $Gviz_dir_uni;
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141
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142 open my $gfai, '>', $fai_file;
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143 foreach my $k ( sort keys %{$fai_ref_hashP} )
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144 {
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145 print $gfai "$k\t$fai_ref_hashP->{$k}\n";
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146 }
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147 close $gfai;
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148 bg_to_png ( $fai_file, $fastq_prefix.'_unique_plus.bedgraph', $fastq_prefix.'_unique_minus.bedgraph', $Gviz_dir_uni, 'Mb' );
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149 bg_to_png ( $fai_file, $fastq_prefix.'_plus.bedgraph', $fastq_prefix.'_minus.bedgraph', $Gviz_dir_rand, 'Mb' );
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150
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151 my $group_dir = $dir_fq.'subgroups/';
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152 my $fastq_uni = $fq_collection.$fastq_n[$child].'_unique_mappers.fastq';
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153 my $fastq_all = $fq_collection.$fastq_n[$child].'_all_mappers.fastq';
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154 my ($bo, $mi, $pi) = subgroups ( $fastq_all, $group_dir, $mis, $misTE, $proc_child, $tRNAs, $rRNAs, $snRNAs, $miRNAs, $transcripts, $TE, $si_min, $si_max, $pi_min, $pi_max, $report);
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155
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156 pie_chart($group_dir);
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157
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158 open (my $dupnum, $gen_dir.'dup_mapnum.txt') || die "cannot open dup_mapnum.txt $!";
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159 my %dupnum_genome;
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160 my $header = <$dupnum>;
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161 while (<$dupnum>)
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162 {
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163 chomp $_;
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164 my @dupline = split /\t/, $_;
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165 $dupnum_genome{$dupline[0]} = [$dupline[1], $dupline[2]];
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166 }
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167 close $dupnum;
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168
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169 my $mi_sam = $group_dir.'miRNAs.sam';
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170 mkdir $group_dir.'miRNAs/';
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171 my $mi_count_file = $group_dir.'miRNAs/miRNAs_reads_counts.txt';
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172 my ( $mi_count, $mi_ref_size ) = sam_count ( $mi_sam );
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173
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174 rpms_rpkm( $mi_count, $mi_ref_size, $ma, $mi_count_file, $pi, $mi, $bo );
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175
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176 my ( $sam_transcripts, $sam_TEs ) = ( $group_dir.'transcripts.sam', $group_dir.'TEs.sam' );
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177 my @types = ($group_dir.'bonafide_reads.fastq', $group_dir.'miRNAs.fastq', $group_dir.'siRNAs.fastq', $group_dir.'piRNAs.fastq' );
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178 my @types_names = ('bonafide_reads', 'miRNAs', 'siRNAs', 'piRNAs');
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179 foreach my $grand_child ( 0 .. $#types )
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180 {
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181 my $type_dir = $group_dir.$types_names[$grand_child].'/';
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182 my $type_prefix = $types_names[$grand_child].'-';
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183 mkdir $type_dir;
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184 $pm2->start($types[$grand_child]) and next;
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185 my ( $type_sam_genome, $type_sam_TEs, $type_sam_transcripts ) = ( $type_dir.$type_prefix.'genome.sam', $type_dir.$type_prefix.'TEs.sam', $type_dir.$type_prefix.'transcripts.sam' );
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186 my ( $type_sam_uni_genome, $type_sam_uni_TEs, $type_sam_uni_transcripts ) = ( $type_dir.$type_prefix.'genome_unique.sam', $type_dir.$type_prefix.'TEs_unique.sam', $type_dir.$type_prefix.'transcripts_unique.sam' );
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187 my ( $type_uni_genome_fastq, $type_uni_TEs_fastq, $type_uni_transcripts_fastq ) = ( $fq_collection.$fastq_n[$child].'-'.$type_prefix.'genome_uni.fastq', $fq_collection.$fastq_n[$child].'-'.$type_prefix.'TEs_uni.fastq', $fq_collection.$fastq_n[$child].'-'.$type_prefix.'transcripts_uni.fastq');
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188 my ( $type_genome_fastq, $type_TEs_fastq, $type_transcripts_fastq ) = ( $fq_collection.$fastq_n[$child].'-'.$type_prefix.'genome.fastq', $fq_collection.$fastq_n[$child].'-'.$type_prefix.'TEs.fastq', $fq_collection.$fastq_n[$child].'-'.$type_prefix.'transcripts.fastq');
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189 my $type_sequence_hashP = get_fastq_seq ( $types[$grand_child] );
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190
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191 if ( $grand_child == 1 )
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192 {
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193 BWA_call ( $TE, $types[$grand_child], $type_sam_TEs, $misTE, $proc_child, $report );
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194 BWA_call ( $transcripts, $types[$grand_child], $type_sam_transcripts, $mis, $proc_child, $report );
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195 BWA_call ( $ref, $types[$grand_child], $type_sam_genome, $mis, $proc_child, $report );
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196 extract_sam ( undef, $type_sam_TEs, $type_sam_TEs, $type_sam_uni_TEs, $type_uni_TEs_fastq, $type_uni_TEs_fastq );
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197 extract_sam ( undef, $type_sam_transcripts, $type_sam_transcripts, $type_sam_uni_transcripts, $type_transcripts_fastq, $type_uni_transcripts_fastq );
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198 extract_sam ( undef, $type_sam_genome, $type_sam_genome, $type_sam_uni_genome, $type_genome_fastq, $type_uni_genome_fastq );
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199 }
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200 else
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201 {
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202 extract_sam ( $type_sequence_hashP, $sam_TEs, $type_sam_TEs, $type_sam_uni_TEs, $type_TEs_fastq, $type_uni_TEs_fastq );
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203 extract_sam ( $type_sequence_hashP, $sam_transcripts, $type_sam_transcripts, $type_sam_uni_transcripts, $type_transcripts_fastq, $type_uni_transcripts_fastq );
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204 extract_sam ( $type_sequence_hashP, $sam_genome, $type_sam_genome, $type_sam_uni_genome, $type_genome_fastq, $type_uni_genome_fastq );
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205 }
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206
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207 my $ex_count_file = $type_dir.$type_prefix.'transcripts_reads_counts.txt';
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208 my ( $ex_count, $ex_ref_size ) = sam_count ( $type_sam_transcripts );
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209 rpms_rpkm( $ex_count, $ex_ref_size, $ma, $ex_count_file, $pi, $mi, $bo );
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210
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211 my ( $TEs_count, $TEs_ref_size, $TEs_count_NoM, $TEs_count_M ) = sam_count_mis ( $type_sam_TEs );
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212 my $TEs_count_file = $type_dir.$type_prefix.'TEs_reads_counts.txt';
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213 my $TEs_count_file_M = $type_dir.$type_prefix.'TEs_reads_counts_mismatches.txt';
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214 my $TEs_count_file_noM = $type_dir.$type_prefix.'TEs_reads_counts_nomismatches.txt';
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21
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215 rpms_rpkm_te( $TEs_count, $TEs_ref_size, $ma, $TEs_count_file, $pi, $mi, $bo );
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216 rpms_rpkm_te( $TEs_count_NoM, $TEs_ref_size, $ma, $TEs_count_file_noM, $pi, $mi, $bo );
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217 rpms_rpkm_te( $TEs_count_M, $TEs_ref_size, $ma, $TEs_count_file_M, $pi, $mi, $bo );
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218
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219 sam_to_bam_bg ( $type_sam_TEs, $scale, $grand_child );
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220 sam_sorted_bam ( $type_sam_transcripts, $grand_child ); sam_sorted_bam ( $type_sam_uni_transcripts, $grand_child );
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221 sam_sorted_bam ( $type_sam_uni_TEs, $grand_child );
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222
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223 my $Gviz_TEs = $type_dir.'Gviz_TEs/';
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224 mkdir $Gviz_TEs;
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225 bg_to_png ( $group_dir.'TEs.fai', $type_dir.$type_prefix.'TEs_plus.bedgraph', $type_dir.$type_prefix.'TEs_minus.bedgraph', $Gviz_TEs, 'Kb' );
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226
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227 my $Gviz_genome= $type_dir.'Gviz_genome/';
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228 my $Gviz_genome_rand = $Gviz_genome.'rand/';
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229 my $Gviz_genome_uni = $Gviz_genome.'unique/';
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230 mkdir $Gviz_genome; mkdir $Gviz_genome_uni; mkdir $Gviz_genome_rand;
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231
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232 sam_to_bam_bg ( $type_sam_genome, $scale, $grand_child );
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233 sam_to_bam_bg ( $type_sam_uni_genome, $scale, $grand_child );
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234
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235 bg_to_png ( $fai_file, $type_dir.$type_prefix.'genome_unique_plus.bedgraph', $type_dir.$type_prefix.'genome_unique_minus.bedgraph', $Gviz_genome_uni, 'Mb' );
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236 bg_to_png ( $fai_file, $type_dir.$type_prefix.'genome_plus.bedgraph', $type_dir.$type_prefix.'genome_minus.bedgraph', $Gviz_genome_rand, 'Mb' );
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237
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238 #HTML Details
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239 my $prefix_details_pages = $dir.$fastq_n[$child].'-'.$types_names[$grand_child];
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240 details_pages ( $type_dir, $prefix_details_pages, \@fastq_n, $fastq_n[$child], $misTE, $dir, $Pcheck );
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241
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242 $pm2->finish();
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243 }
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244 $pm2->wait_all_children;
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245
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246 if ( $Pcheck eq 'true' )
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247 {
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248 my $ppp = $group_dir.'PPPartners/'; mkdir $ppp;
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249 print $report "ping_pong_partners $group_dir/piRNAs/TEs.sam $ppp\n";
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250 ping_pong_partners ( $group_dir.'TEs.fai', $group_dir.'piRNAs/piRNAs-TEs_sorted.bam', $ppp, $pi_min );
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251 my $ppp_page = $dir.$fastq_n[$child].'-piRNAs-PPP.html';
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252 ppp_page ( $group_dir, $ppp_page, \@fastq_n, $fastq_n[$child], $ppp, $dir );
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253 }
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254
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255 #HTML Main Webpage
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256 my $index_page = $dir.$fastq_n[$child].'.html';
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257 main_page ( $gen_dir, $index_page, \@fastq_n, $fastq_n[$child], $ma, $ma_uni, $dir );
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258 copy ($index_page, $html_out) if $child == 0;
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259 #HTML Menu
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260 my $menu_page = $dir.$fastq_n[$child].'-sub.html';
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261 menu_page ( $group_dir, $menu_page, \@fastq_n, $fastq_n[$child], $min, $max, $si_min, $si_max, $pi_min, $pi_max, $dir );
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262 unlink glob "$group_dir*.sam"; unlink glob "$group_dir*.fastq";
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263 $pm->finish(); # pass an exit code to finish
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264 }
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265 $pm->wait_all_children;
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266 unlink glob $dir."dataset_*symlink.fa*";
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267 print $report "Job done!\n";
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268 close $report;
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