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1 package align;
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2
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3 use strict;
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4 use warnings;
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5 use File::Basename;
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6 use String::Random;
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7
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8 use FindBin;
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9 use lib $FindBin::Bin;
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10 use Rcall qw ( histogram );
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11
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12 use Exporter;
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13 our @ISA = qw( Exporter );
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20
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14 our @EXPORT = qw( &rpms_rpkm_te &BWA_call &to_build &get_unique &sam_sorted_bam &get_hash_alignment &sam_to_bam_bg &sam_count &sam_count_mis &rpms_rpkm &get_fastq_seq &extract_sam );
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1
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15
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16 sub to_build
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17 {
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18 my ( $toBuildTabP, $log, $newdir ) = @_;
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19
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20 foreach my $pairs ( @{ $toBuildTabP } )
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21 {
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22 if ( $pairs->[0] == 1 )
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23 {
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24 my $sym = $newdir.basename(${$pairs->[1]}).'_symlink.fa';
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25 symlink( ${$pairs->[1]}, $sym );
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26 ${$pairs->[1]} = $sym;
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27 build_index ( ${$pairs->[1]}, $log );
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28 }
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29 }
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30 }
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31
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32 sub build_index
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33 {
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34 my $to_index = shift;
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35 my $log = shift;
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36 my $index_log = $to_index.'_index.err';
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37 `bwa index $to_index 2> $index_log`;
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38 print $log "Creating index for $to_index\n";
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39 }
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40
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41 sub get_unique
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42 {
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43 my ( $sam, $s_uni, $out_prefix, $col_prefix, $details, $report ) = @_;
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44
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45 my $fout = $col_prefix.'_all_mappers.fastq';
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46 my $funi = $col_prefix.'_unique_mappers.fastq';
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47 my $frej = $col_prefix.'_unmapped.fastq';
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48
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49 my $repartition = $out_prefix.'distribution.txt';
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50 my $png_rep = $out_prefix.'distribution.png';
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51 my ( %duplicates, %genome_hits) ;
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52
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53 #alignement to the first reference
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54 my @return = sam_parse( $sam, $fout, $funi, $frej, $s_uni, \%duplicates, \%genome_hits, $report );
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55 my $ref_fai = $return[4];
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56 my $mappers = $return[5];
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57 my $mappers_uni = $return[6];
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58 my $size_mappedHashR = $return[7];
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59
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60 if ( $details == 1 )
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61 {
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62 #print number of duplicates and hits number
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63 my ($pourcentage, $total) =(0,0);
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64
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65 $total += $_ foreach values %{$size_mappedHashR};
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66 open (my $rep, '>'.$repartition) || die "cannot create $repartition $!\n";
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67 print $rep "size\tnumber\tpercentage\n";
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68 foreach my $k (sort{$a cmp $b} keys (%{$size_mappedHashR}))
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69 {
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70 $pourcentage = 0;
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71 $pourcentage = $size_mappedHashR->{$k} / $total * 100 unless $total ==0;
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72
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73 print $rep "$k\t$size_mappedHashR->{$k}\t";
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74 printf $rep "%.2f\n",$pourcentage;
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75 }
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76
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77 histogram($size_mappedHashR, $png_rep, $total);
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78
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79
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80 my $dup = $out_prefix.'dup_mapnum.txt';
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81 my $dup_u = $out_prefix .'dup_unique.txt';
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82 my $dup_r = $out_prefix .'dup_nonmapp.txt';
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83 open(my $tab,">".$dup) || die "cannot open output txt file\n";
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84 open(my $tab_r,">".$dup_r) || die "cannot open output txt file\n";
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85 open(my $tab_u,">".$dup_u) || die "cannot open output txt file\n";
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86 print $tab "sequence\tcount\tmapnum\n";
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87 print $tab_u "sequence\tcount\n";
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88 print $tab_r "sequence\tcount\n";
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89 foreach my $k (sort {$duplicates{$b} <=> $duplicates{$a}}keys %duplicates)
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90 {
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91 $duplicates{$k} = 0 unless exists($duplicates{$k});
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92 $genome_hits{$k} = 0 unless exists($genome_hits{$k});
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93 if ($genome_hits{$k} != 0) { print $tab $k."\t".$duplicates{$k}."\t".$genome_hits{$k}."\n"; }
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94 else {print $tab_r $k."\t".$duplicates{$k}."\n";}
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95 if ($genome_hits{$k} == 1) { print $tab_u $k."\t".$duplicates{$k}."\n"; }
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96 }
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97 close $dup; close $dup_r; close $dup_u;
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98 }
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99 return ( $ref_fai, $mappers, $mappers_uni );
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100 }
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101
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102 sub sam_parse
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103 {
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104 my ( $sam, $fastq_accepted, $fastq_accepted_unique, $fastq_rejected, $sam_unique, $duplicate_hashR, $best_hit_number_hashR, $report ) = @_ ;
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105 my ($reads, $mappers, $mappersUnique, @garbage, %size_num, %size_num_spe, %number, %numberSens, %numberReverse, %unique_number, %numberNM, %numberM, %size);
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106 $mappers = $mappersUnique = $reads = 0;
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107
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108 open my $fic, '<', $sam || die "cannot open $sam $!\n";
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109 open my $accepted, '>', $fastq_accepted || die "cannot create $fastq_accepted $! \n";
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110 open my $unique, '>', $fastq_accepted_unique || die "cannot create $fastq_accepted_unique $! \n";
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111 open my $rejected, '>', $fastq_rejected || die "cannot create $fastq_rejected $! \n";
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112 open my $sam_uni, '>', $sam_unique || die "cannot create $sam_unique $! \n";
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113 my $sequence = '';
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114 while(<$fic>)
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115 {
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116 chomp $_;
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117 if ($_ =~ /^\@[A-Za-z][A-Za-z](\t[A-Za-z][A-Za-z0-9]:[ -~]+)+$/ || $_ =~ /^\@CO\t.*/ )
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118 {
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119 if ($_ =~ /\@SQ\tSN:(.*)\tLN:(\d*)/)
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120 {
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121 $size{$1} = $2;
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122 $unique_number{$1} = 0;
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123 $number{$1} = 0;
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124 $numberNM{$1} = 0;
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125 $numberM{$1} = 0;
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126 }
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127 print $sam_uni $_."\n";
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128 next;
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129 }
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130 $reads++;
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131 my @line = split (/\t/,$_);
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132 $sequence = $line[9];
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133 if ($line[1] & 16)
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134 {
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135 $sequence =reverse($sequence);
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136 $sequence =~ tr/atgcuATGCU/tacgaTACGA/;
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137 }
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138 if ($line[1] == 16 || $line[1] == 0)
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139 {
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140 my $len = length($sequence);
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141 $size_num{$len} ++;
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142 $size_num_spe{$line[2]}{$len}++;
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143 $mappers ++;
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144
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145 ${$best_hit_number_hashR}{$sequence} = $1 if ($line[13] =~ /X0:i:(\d*)/ || $line[14] =~/X0:i:(\d*)/ );
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146 ${$duplicate_hashR}{$sequence}++;
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147 $number{$line[2]}++;
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148 $numberSens{$line[2]}++ if $line[1] == 0 ;
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149 $numberReverse{$line[2]}++ if $line[1] == 16 ;
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150 print $accepted "\@".$line[0]."\n".$sequence."\n+\n".$line[10]."\n";
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151
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152 if ($line[11] eq "XT:A:U")
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153 {
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154 $unique_number{$line[2]}++;
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155 $mappersUnique++;
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156 print $unique "\@".$line[0]."\n".$sequence."\n+\n".$line[10]."\n";
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157 print $sam_uni $_."\n";
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158 }
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159 if ($_ =~ /.*XM:i:(\d+).*/)
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160 {
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161 if ($1 == 0){$numberNM{$line[2]}++;}else{$numberM{$line[2]}++;}
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162 }
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163 }
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164 else
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165 {
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166 ${$best_hit_number_hashR}{$sequence} = 0;
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167 ${$duplicate_hashR}{$sequence}++;
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168 print $rejected "\@".$line[0]."\n".$sequence."\n+\n".$line[10]."\n";
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169 }
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170 }
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171 close $fic; close $accepted; close $unique; close $rejected; close $sam_uni;
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172
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173 print $report "Parsing $sam file\n";
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174 print $report "\treads: $reads\n";
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175 print $report "\tmappers: $mappers\n";
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176 print $report "\tunique mappers: $mappersUnique\n";
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177 print $report "-----------------------------\n";
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178 return (\%number, \%unique_number, \%numberSens, \%numberReverse, \%size, $mappers, $mappersUnique, \%size_num, \%size_num_spe, \%numberNM, \%numberM );
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179 }
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180
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181 sub get_hash_alignment
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182 {
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183 my ($index, $mismatches, $accept, $reject, $outA, $outR, $fastq, $number_of_cpus, $name, $sam, $report, $fai_f) = @_ ;
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184 my ($reads, $mappers, $unmapped) = (0,0,0);
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185 my $accep_unique;
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186 BWA_call ( $index, $fastq, $sam, $mismatches, $number_of_cpus, $report );
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187
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188 open my $fic, '<', $sam || die "cannot open $sam $!\n";
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189 open my $accepted, '>', $outA || die "cannot open $outA\n" if $accept == 1;
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190 open my $rejected, '>', $outR || die "cannot open $outR\n" if $reject == 1;
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191 open my $fai, '>', $fai_f || die "cannot open $fai_f\n" if $fai_f;
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192 #if ($name eq "snRNAs") {
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193 # open ( $accep_unique, ">".$1."-unique.fastq") if $outR =~ /(.*)\.fastq/;
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194 #}
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195 my $sequence = '';
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196 while(<$fic>)
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197 {
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198 chomp $_;
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199 if( $_ =~ /^\@[A-Za-z][A-Za-z](\t[A-Za-z][A-Za-z0-9]:[ -~]+)+$/ || $_ =~ /^\@CO\t.*/ )
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200 {
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201 if ($fai_f && $_ =~ /\@SQ\tSN:(.*)\tLN:(\d*)/)
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202 {
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203 print $fai $1."\t".$2."\n";
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204 }
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205 next;
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206 }
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207 $reads++;
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208 my @line = split (/\t/,$_);
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209 $sequence = $line[9];
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210 if ($line[1] & 16)
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211 {
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212 $sequence =reverse($sequence);
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213 $sequence =~ tr/atgcuATGCU/tacgaTACGA/;
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214 }
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215 if ($line[1] & 16 || $line[1] == 0)
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216 {
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217 $mappers ++;
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218 if ($accept == 1 )
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219 {
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220 print $accepted "\@".$line[0]."\n".$sequence."\n+\n".$line[10]."\n";
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221 # print $accep_unique "\@".$line[0]."\n".$sequence."\n+\n".$line[10]."\n" if ($name eq "snRNAs" && $line[11] eq "XT:A:U");
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222 }
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223 }
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224 else
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225 {
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226 print $rejected "\@".$line[0]."\n".$sequence."\n+\n".$line[10]."\n" if $reject == 1;
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227 $unmapped++;
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228 }
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229 }
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230 # close $accep_unique if ($name eq "bonafide_reads");
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231 close $fic;
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232 close $accepted if $accept == 1;
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233 close $rejected if $reject ==1;
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234 close $fai if $fai_f;
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235 print $report "\treads: $reads\n";
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236 print $report "\tmappers: $mappers\n";
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237 print $report "\tunmapped: $unmapped\n";
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238 print $report "-----------------------------\n";
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239 return ($mappers, $unmapped);
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240 }
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241
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242 sub sam_count
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243 {
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244 my $sam = shift;
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245 my ( %number, %size );
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246
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247 open my $fic, '<', $sam || die "cannot open $sam file $!\n";
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248 while(<$fic>)
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249 {
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250 chomp $_;
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251 if ($_ =~ /^\@[A-Za-z][A-Za-z](\t[A-Za-z][A-Za-z0-9]:[ -~]+)+$/ || $_ =~ /^\@CO\t.*/ )
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252 {
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253 if ($_ =~ /\@SQ\tSN:(.*)\tLN:(\d*)/)
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254 {
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255 $size{$1} = $2;
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256 $number{$1} = 0;
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257 }
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258 }
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259 else
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260 {
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261 my @line = split (/\t/,$_);
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262 if ( $line[1] & 16 || $line[1] == 0 )
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263 {
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264 $number{$line[2]}++;
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265 }
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266 }
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267 }
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268 close $fic;
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269 return ( \%number, \%size );
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270 }
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271
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272 sub sam_count_mis
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273 {
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274
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275 my $sam = shift;
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276 my ( %number, %numberNM, %numberM, %size);
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277
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278 open my $fic, '<', $sam || die "cannot open $sam file $!\n";
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279 while(<$fic>)
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280 {
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281 chomp $_;
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282 if ($_ =~ /^\@[A-Za-z][A-Za-z](\t[A-Za-z][A-Za-z0-9]:[ -~]+)+$/ || $_ =~ /^\@CO\t.*/ )
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283 {
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284 if ($_ =~ /\@SQ\tSN:(.*)\tLN:(\d*)/)
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285 {
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286 $size{$1} = $2;
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20
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287 $number{$1} = [0,0,0,0,0];
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288 $numberNM{$1} = [0,0,0,0,0];
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289 $numberM{$1} = [0,0,0,0,0];
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1
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290 }
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291 }
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292 else
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293 {
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294 my @line = split (/\t/,$_);
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20
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295 my @seq = split //, $line[9];
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296 if ( $line[1] == 16 || $line[1] == 0 )
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1
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297 {
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20
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298 $number{ $line[2] }->[0]++;
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299 if ($line[1] == 0)
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300 {
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301 $number{$line[2]}->[1]++;
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302 $number{$line[2]}->[3]++ if $seq[0] eq 'T';
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303 }
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304 else
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305 {
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25
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306 $number{$line[2]}->[2]++;
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24
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307 $number{$line[2]}->[4]++ if $seq[9] eq 'A';
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20
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308 }
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1
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309 if ($_ =~ /.*XM:i:(\d+).*/)
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310 {
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20
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311 if ( $1 == 0 )
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312 {
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313 $numberNM{$line[2]}->[0]++;
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314 if ($line[1] == 0)
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315 {
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316 $numberNM{$line[2]}->[1]++;
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317 $numberNM{$line[2]}->[3]++ if $seq[0] eq 'T';
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318 }
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319 else
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320 {
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321 $numberNM{$line[2]}->[2]++;
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322 $numberNM{$line[2]}->[4]++ if $seq[9] eq 'A';
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323 }
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324 }
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325 else
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326 {
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327 $numberM{$line[2]}->[0]++;
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328 if ($line[1] == 0)
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329 {
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330 $numberM{$line[2]}->[1]++;
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331 $numberM{$line[2]}->[3]++ if $seq[0] eq 'T';
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332 }
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333 else
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334 {
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335 $numberM{$line[2]}->[2]++;
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22
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336 $numberM{$line[2]}->[4]++ if $seq[9] eq 'A';
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20
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337 }
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338 }
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1
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339 }
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340 }
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341 }
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342 }
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343 return (\%number, \%size, \%numberNM, \%numberM );
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344 }
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345
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20
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346 sub rpms_rpkm_te
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347 {
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348 my ( $counthashR, $sizehashR, $mapped, $out_file, $piRNA_number, $miRNA_number, $bonafide_number ) =@_;
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349 open(my $out, ">".$out_file) || die "cannot open normalized file $! \n";
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350 print $out "ID\treads counts\tRPKM\tsens reads counts\treverse reads counts\t% of sens 1U\t% of reverse 10A";
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351 print $out "\tper million of piRNAs" if ($piRNA_number != 0);
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352 print $out "\tper million of miRNAs" if ($miRNA_number != 0);
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353 print $out "\tper million of bonafide reads" if ($bonafide_number != 0);
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354 print $out "\n";
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355 foreach my $k ( sort keys %{$counthashR} )
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356 {
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357 my ($rpkm, $pirna, $mirna, $bonafide) = (0,0,0,0);
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358
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359 $rpkm = ( $counthashR->{$k}->[0] * 1000000000) / ( $sizehashR->{$k} * $mapped) if ( $sizehashR->{$k} * $mapped) != 0 ;
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360 print $out $k."\t".$counthashR->{$k}->[0]."\t"; printf $out "%.2f",$rpkm;
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361 print $out "\t".$counthashR->{$k}->[1]."\t".$counthashR->{$k}->[2] ;
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362 my $U1 = 0;
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363 $U1 = $counthashR->{$k}->[3] / $counthashR->{$k}->[1] * 100 if $counthashR->{$k}->[1] != 0;
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364 my $A10 = 0;
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365 $A10 = $counthashR->{$k}->[4] / $counthashR->{$k}->[2] * 100 if $counthashR->{$k}->[2] != 0;
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366
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367 print $out "\t".$U1."\t".$A10;
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368
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369 if ($piRNA_number != 0 )
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370 {
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371 $pirna = ( $counthashR->{$k}->[0] * 1000000) / $piRNA_number;
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372 printf $out "\t%.2f",$pirna;
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373 }
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374 if ($miRNA_number != 0 )
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375 {
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376 $mirna = ( $counthashR->{$k}->[0] * 1000000) / $miRNA_number;
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377 printf $out "\t%.2f",$mirna;
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378 }
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379 if ($bonafide_number != 0 )
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380 {
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381 $bonafide = ( $counthashR->{$k}->[0] * 1000000) / $bonafide_number;
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382 printf $out "\t%.2f",$bonafide;
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383 }
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384 print $out "\n";
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385 }
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386 close $out;
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387 }
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388
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389
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1
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390 sub sam_to_bam_bg
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391 {
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392 my ( $sam, $scale, $number_of_cpus ) = @_;
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393 my ( $bam_sorted, $bedgraphM, $bedgraphP, $view_err, $sort_err ) = ( '', '', '', '', '' );
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394 if ( $sam =~ /(.*?).sam$/ )
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395 {
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396 $bam_sorted = $1.'_sorted.bam';
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397 $bedgraphP= $1.'_plus.bedgraph';
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398 $bedgraphM = $1.'_minus.bedgraph';
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399 $view_err = $1.'_view.err';
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400 $sort_err = $1.'_sort.err';
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401 }
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402 `samtools view -Shb --threads $number_of_cpus $sam 2> $view_err | samtools sort -O BAM --threads $number_of_cpus /dev/stdin 2> $sort_err > $bam_sorted`;
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403 `bedtools genomecov -scale $scale -strand + -bga -ibam $bam_sorted > $bedgraphP`;
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404 `bedtools genomecov -scale $scale -strand - -bga -ibam $bam_sorted > $bedgraphM`;
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405 }
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406
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407 sub sam_sorted_bam
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408 {
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409 my ( $sam, $number_of_cpus ) = @_;
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410 my ( $bam_sorted, $view_err, $sort_err ) = ( '', '', '' );
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411 if ( $sam =~ /(.*?).sam$/ )
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412 {
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413 $bam_sorted = $1.'_sorted.bam';
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414 $view_err = $1.'_view.err';
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415 $sort_err = $1.'_sort.err';
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416
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417 }
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418 `samtools view -Shb --threads $number_of_cpus $sam 2> $view_err | samtools sort -O BAM --threads $number_of_cpus /dev/stdin 2> $sort_err > $bam_sorted`;
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419 }
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420
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421 sub BWA_call
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422 {
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423 my ( $index, $fastq, $sam, $mismatches, $number_of_cpus, $report ) = @_;
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424 my ( $aln_err, $samse_err, $seq_num ) = ( $sam.'_aln.err', $sam.'_samse.err', 0 );
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425 print $report "-----------------------------\n";
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426 print $report "bwa aln -t $number_of_cpus -n $mismatches $index $fastq 2> $aln_err | bwa samse $index /dev/stdin $fastq 2> $samse_err > $sam\n";
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427 `bwa aln -t $number_of_cpus -n $mismatches $index $fastq 2> $aln_err | bwa samse $index /dev/stdin $fastq 2> $samse_err > $sam `;
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428 }
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429
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430 sub rpms_rpkm
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431 {
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432 my ( $counthashR, $sizehashR, $mapped, $out_file, $piRNA_number, $miRNA_number, $bonafide_number ) =@_;
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433 open(my $out, ">".$out_file) || die "cannot open normalized file $! \n";
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434 print $out "ID\treads counts\tRPKM";
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435 print $out "\tper million of piRNAs" if ($piRNA_number != 0);
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436 print $out "\tper million of miRNAs" if ($miRNA_number != 0);
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437 print $out "\tper million of bonafide reads" if ($bonafide_number != 0);
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438 print $out "\n";
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439 foreach my $k ( sort keys %{$counthashR} )
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440 {
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441 my ($rpkm, $pirna, $mirna, $bonafide) = (0,0,0,0);
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442
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443 $rpkm = ( $counthashR->{$k} * 1000000000) / ( $sizehashR->{$k} * $mapped) if ( $sizehashR->{$k} * $mapped) != 0 ;
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444 print $out $k."\t".$counthashR->{$k}."\t"; printf $out "%.2f",$rpkm;
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|
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445
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|
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446 if ($piRNA_number != 0 )
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447 {
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448 $pirna = ( $counthashR->{$k} * 1000000) / $piRNA_number;
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|
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449 printf $out "\t%.2f",$pirna;
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|
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450 }
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|
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451 if ($miRNA_number != 0 )
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|
|
452 {
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|
|
453 $mirna = ( $counthashR->{$k} * 1000000) / $miRNA_number;
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|
|
454 printf $out "\t%.2f",$mirna;
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|
|
455 }
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|
|
456 if ($bonafide_number != 0 )
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|
|
457 {
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|
|
458 $bonafide = ( $counthashR->{$k} * 1000000) / $bonafide_number;
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|
|
459 printf $out "\t%.2f",$bonafide;
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|
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460 }
|
|
|
461 print $out "\n";
|
|
|
462 }
|
|
|
463 close $out;
|
|
|
464 }
|
|
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465
|
|
|
466 sub extract_sam
|
|
|
467 {
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|
|
468 my ( $hashRef, $sam_in, $sam_out, $sam_uni_out, $fastq_out, $fastq_uni_out ) = @_;
|
|
|
469
|
|
|
470 open my $s_in, '<', $sam_in || die "cannot open $sam_in file $!\n";
|
|
|
471
|
|
|
472 open my $f_out, '>', $fastq_out || die "cannot create $fastq_out $!\n";
|
|
|
473 open my $f_uni_out, '>', $fastq_uni_out || die "cannot create $fastq_uni_out $!\n";
|
|
|
474
|
|
|
475 open my $s_out, '>', $sam_out || die "cannot create $sam_out file $!\n" if defined ($hashRef);
|
|
|
476 open my $s_uni_out, '>', $sam_uni_out || die "cannot create $sam_uni_out file $!\n";
|
|
|
477
|
|
|
478 my $sequence = '';
|
|
|
479 while(<$s_in>)
|
|
|
480 {
|
|
|
481 if ($_ =~ /^\@[A-Za-z][A-Za-z](\t[A-Za-z][A-Za-z0-9]:[ -~]+)+$/ || $_ =~ /^\@CO\t.*/ )
|
|
|
482 {
|
|
|
483 print $s_out $_ if defined ($hashRef);
|
|
|
484 print $s_uni_out $_;
|
|
|
485 next;
|
|
|
486 }
|
|
|
487 my @line = split (/\t/,$_);
|
|
|
488 $sequence = $line[0];
|
|
|
489 if ( (! defined ($hashRef) )|| ( exists $hashRef->{$sequence} && $hashRef->{$sequence} == 1 ) )
|
|
|
490 {
|
|
|
491 my $arn = $line[9];
|
|
|
492 if ($line[1] & 16)
|
|
|
493 {
|
|
|
494 $arn =reverse($arn);
|
|
|
495 $arn =~ tr/atgcuATGCU/tacgaTACGA/;
|
|
|
496 }
|
|
|
497
|
|
|
498 if ( ( $line[1] == 16 || $line[1] == 0 ) )
|
|
|
499 {
|
|
|
500 print $f_out "\@".$line[0]."\n".$arn."\n+\n".$line[10]."\n" ;
|
|
|
501 print $s_out $_ if defined ($hashRef);
|
|
|
502 if ( $line[11] eq "XT:A:U" )
|
|
|
503 {
|
|
|
504 print $f_uni_out "\@".$line[0]."\n".$arn."\n+\n".$line[10]."\n" ;
|
|
|
505 print $s_uni_out $_ ;
|
|
|
506 }
|
|
|
507 }
|
|
|
508 }
|
|
|
509 }
|
|
|
510 close $s_in; close $s_out if defined ($hashRef);
|
|
|
511 close $s_uni_out; close $f_out; close $f_uni_out;
|
|
|
512 }
|
|
|
513
|
|
|
514 sub get_fastq_seq
|
|
|
515 {
|
|
|
516 my $fastq = shift;
|
|
|
517 my %hash; my $cmp = 0;
|
|
|
518
|
|
|
519 open my $fic, '<', $fastq || die "cannot open input file $! \n";
|
|
|
520 while(<$fic>)
|
|
|
521 {
|
|
|
522 chomp $_;
|
|
|
523 $cmp++;
|
|
|
524 if ($cmp % 4 == 1)
|
|
|
525 {
|
|
|
526 die "file do not contain a @ at line $cmp\n" unless ($_ =~ /^\@/ );
|
|
|
527 if ($_ =~ /^\@(.*)\s.*/) { $hash{$1} = 1;}
|
|
|
528 elsif ($_ =~ /^\@(.*)/) { $hash{$1} = 1;}
|
|
|
529 }
|
|
|
530 elsif ($cmp % 4 == 3 )
|
|
|
531 {
|
|
|
532 die "file do not contain a + at line $cmp\n" unless $_ =~ /^\+/;
|
|
|
533 }
|
|
|
534 }
|
|
|
535 close $fic;
|
|
|
536 return \%hash;
|
|
|
537 }
|
|
|
538
|
|
|
539 1;
|
