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1 #!/usr/bin/perl
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2
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3 ####
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4 #### Usage: SoftSearch.pl [-lqrmsd] -b <BAM> -f <Genome.fa> -sam <samtools path> -bed <bedtools path>
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5 #### Created 1-30-2012 by Steven Hart, PhD
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6 #### hart.steven@mayo.edu
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7 #### Required bedtools & samtools to be in path
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8
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9
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10 use lib "/data2/bsi/reference/softsearch/lib" ;
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11
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12 use Getopt::Long;
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13 use strict;
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14 use warnings;
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15 #use Data::Dumper;
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16 use LevD;
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17 use File::Basename;
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18
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19 my ($INPUT_BAM,$INPUT_FASTA,$OUTPUT_FILE,$minSoft,$minSoftReads,$dist_To_Soft,$bedtools,$samtools);
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20 my ($minRP, $temp_output, $num_sd, $MapQ, $chrom, $unmated_pairs, $minBQ, $pair_only, $disable_RP_only);
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21 my ($levD_local_threshold, $levD_distl_threshold,$pe_upper_limit,$high_qual,$sv_only,$blacklist,$genome_file,$verbose);
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22
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23 my $cmd = "";
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24
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25 #Declare variables
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26 GetOptions(
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27 'b=s' => \$INPUT_BAM,
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28 'f=s' => \$INPUT_FASTA,
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29 'o:s' => \$OUTPUT_FILE,
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30 'm:i' => \$minRP,
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31 'l:i' => \$minSoft,
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32 'r:i' => \$minSoftReads,
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33 't:i' => \$temp_output,
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34 's:s' => \$num_sd,
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35 'd:i' => \$dist_To_Soft,
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36 'q:i' => \$MapQ,
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37 'c:s' => \$chrom,
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38 'u:s' => \$unmated_pairs,
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39 'x:s' => \$minBQ,
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40 'p' => \$pair_only,
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41 'g' => \$disable_RP_only,
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42 'j:s' => \$levD_local_threshold,
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43 'k:s' => \$levD_distl_threshold,
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44 'a:s' => \$pe_upper_limit,
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45 'e:s' => \$high_qual,
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46 'L' => \$sv_only,
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47 'v' => \$verbose,
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48 'blacklist:s' => \$blacklist,
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49 'genome_file:s' => \$genome_file,
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50 "help|h|?" => \&usage);
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51
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52 unless($sv_only){$sv_only=""};
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53 if(defined($INPUT_BAM)){$INPUT_BAM=$INPUT_BAM} else {print usage();die "Where is the BAM file?\n\n"}
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54 if(defined($INPUT_FASTA)){$INPUT_FASTA=$INPUT_FASTA} else {print usage();die "Where is the fasta file?\n\n"}
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55 my ($fn,$pathname) = fileparse($INPUT_BAM,".bam");
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56 my $index=`ls $pathname/$fn*bai|head -1`;
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57 #my $index =`ls \${INPUT_BAM%.bam}*bai`;
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58 #print "INDEX=$index\n";
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59 if(!$index){die "\n\nERROR: you need index your BAM file\n\n"}
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60
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61 ### get current time
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62 print "Start Time : " . &spGetCurDateTime() . "\n";
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63 my $now = time;
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64
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65 #if(defined($OUTPUT_FILE)){$OUTPUT_FILE=$OUTPUT_FILE} else {$OUTPUT_FILE="output.vcf"; print "\nNo outfile specified. Using output.vcf as default\n\n"}
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66 if(defined($minSoft)){$minSoft=$minSoft} else {$minSoft=5}
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67 if(defined($minRP)){$minRP=$minRP} else {$minRP=5}
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68 if(defined($minSoftReads)){$minSoftReads=$minSoftReads} else {$minSoftReads=5}
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69 if(defined($dist_To_Soft)){$dist_To_Soft=$dist_To_Soft} else {$dist_To_Soft=300}
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70 if(defined($num_sd)){$num_sd=$num_sd} else {$num_sd=6}
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71 if(defined($MapQ)){$MapQ=$MapQ} else {$MapQ=20}
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72
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73 unless (defined $pe_upper_limit) { $pe_upper_limit = 10000; }
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74 unless (defined $levD_local_threshold) { $levD_local_threshold = 0.05; }
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75 unless (defined $levD_distl_threshold) { $levD_distl_threshold = 0.05; }
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76 #Get sample name if available
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77 my $SAMPLE_NAME="";
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78 my $OUTNAME ="";
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79 $SAMPLE_NAME=`samtools view -f2 -H $INPUT_BAM|awk '{if(\$1~/^\@RG/){sub("ID:","",\$2);name=\$2;print name}}'|head -1`;
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80 $SAMPLE_NAME=~s/\n//g;
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81 if (!$OUTPUT_FILE){
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82 if($SAMPLE_NAME ne ""){$OUTNAME=$SAMPLE_NAME.".vcf"}
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83 else {$OUTNAME="output.vcf"}
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84 }
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85 else{$OUTNAME=$OUTPUT_FILE}
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86
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87 print "Writing results to $OUTNAME\n";
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88
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89
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90 ##Make sure if submitting on SGE, to prepned the "chr". Not all referecne FAST files require "chr", so we shouldn't force the issue.
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91 if(!defined($chrom)){$chrom=""}
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92 if(!defined($unmated_pairs)){$unmated_pairs=0}
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93
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94 my $badQualValue=chr($MapQ);
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95 if(defined($minBQ)){ $badQualValue=chr($minBQ); }
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96
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97 if($badQualValue eq "#"){$badQualValue="\#"}
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98
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99 # adding and cheking for samtools and bedtools in the PATh
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100 ## check for bedtools and samtools in the path
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101 $bedtools=`which intersectBed` ;
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102 if(!defined($bedtools)){die "\nError:\n\tno bedtools. Please install bedtools and add to the path\n";}
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103 #$samtools=`samtools 2>&1`;
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104 $samtools=`which samtools`;
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105 if($samtools !~ /(samtools)/i){die "\nError:\n\tno samtools. Please install samtools and add to the path\n";}
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106
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107 print "Usage = SoftSearch.pl -l $minSoft -q $MapQ -r $minSoftReads -d $dist_To_Soft -m $minRP -s $num_sd -c $chrom -b $INPUT_BAM -f $INPUT_FASTA -o $OUTNAME \n\n";
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108 sub usage {
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109 print "\nusage: SoftSearch.pl [-cqlrmsd] -b <BAM> -f <Genome.fa> \n";
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110 print "\t-q\t\tMinimum mapping quality [20]\n";
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111 print "\t-l\t\tMinimum length of soft-clipped segment [5]\n";
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112 print "\t-r\t\tMinimum depth of soft-clipped reads at position [5]\n";
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113 print "\t-m\t\tMinimum number of discordant read pairs [5]\n";
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114 print "\t-s\t\tNumber of sd away from mean to be considered discordant [6]\n";
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115 print "\t-u\t\tNumber of unmated pairs [0]\n";
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116 print "\t-d\t\tMax distance between soft-clipped segments and discordant read pairs [300]\n";
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117 print "\t-o\t\tOutput file name [output.vcf]\n";
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118 print "\t-t\t\tPrint temp files for debugging [no|yes]\n";
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119 print "\t-c\t\tuse only this chrom or chr:pos1-pos2\n";
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120 print "\t-p\t\tuse paired-end mode only. In other words, don't try to find soft-clipping events!\n";
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121 print "\t-g\t\tEnable paired-only seach. This will look for discordant read pairs even without soft clips.\n";
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122 print "\t-a\t\tset the minimum distance for a discordant read pair without soft-clipping info [10000]\n";
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123 print "\t-L\t\tFlag to print out even small deletions (low quality)\n";
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124 print "\t-e\t\tdisable strict quality filtering of base qualities in soft-clipped reads [no]\n";
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125 print "\t-blacklist\tareas of the genome to skip calling. Requires -genome_file\n";
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126 print "\t-genome_file\ttab seperated value of chromosome name and length. Only used with -blacklist option\n\n";
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127
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128 exit 1;
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129 }
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130
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131
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132 #############################################################
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133 # create temporary variable name
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134 #############################################################
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135 srand (time ^ $$ ^ unpack "%L*", `ps axww | gzip -f`);
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136 our $random_name=join "", map { ("a".."z")[rand 26] } 1..8;
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137
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138 #############################################################
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139 ## create green list
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140 ##############################################################
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141 #
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142 my $new_blacklist="";
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143 if($blacklist){
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144 if(!$genome_file){die "if using a blacklist, you must also specify the location of a genome_file
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145 The format of the genome_file should be
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146 chrom size
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147 chr1 249250621
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148 chr2 243199373
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149 ...
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150
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151 If using hg19, you can ge the genome file by
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152 mysql --user=genome --host=genome-mysql.cse.ucsc.edu -A -e \"select chrom, size from hg19.chromInfo\" > hg19.genome";}
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153
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154 $cmd=join("","complementBed -i $blacklist -g $genome_file >",$random_name,".bed") ;
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155 system ($cmd);
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156 $new_blacklist=join(""," -L ",$random_name,".bed ");
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157 }
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158
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159 if($verbose){print "CMD=$cmd\nBlacklist is $new_blacklist\n";}
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160
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161
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162
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163
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164
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165 #############################################################
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166 # Calcualte insert size distribution of properly mated reads
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167 #############################################################
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168
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169 #Change for compatability with other operating systems
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170 #my $metrics=`samtools view -q $MapQ -f2 $INPUT_BAM $chrom|cut -f9|head -10000|awk '{if (\$1<0){\$1=-\$1}else{\$1=\$1} sum+=\$1; sumsq+=\$1*\$1} END {print sum/NR, sqrt(sumsq/NR - (sum/NR)**2)}'`;
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171
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172 my $metrics=`samtools view -q $MapQ -f2 $INPUT_BAM $chrom|cut -f9|head -10000|awk '{if (\$1<0){\$1=-\$1}else{\$1=\$1} sum+=\$1; sumsq+=\$1*\$1} END {print sum/NR, sqrt(sumsq/NR - (sum/NR)^2)}'`;
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173 #my ($mean,$stdev)=split(/ /,$metrics);
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174 my ($mean,$stdev)=split(/\s/,$metrics);
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175 $stdev=~s/\n//;
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176 my $upper_limit=int($mean+($num_sd*$stdev));
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177 my $lower_limit=int($mean-($num_sd*$stdev));
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178 die if (!$mean);
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179 print qq{The mean insert size is $mean +/- $stdev (sd)
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180 The upper limit = $upper_limit
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181 The lower limit = $lower_limit\n
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182 };
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183 if($lower_limit<0){
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184 print "Warning!! Given this insert size distribution, we can not call small indels. No other data will be affected\n";
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185 $lower_limit=1;
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186 }
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187 my $tmp_name=join ("",$random_name,".tmp.bam");
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188 my $random_file_sc = "";
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189 my $command = "";
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190
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191 #############################################################
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192 # Make sam file that has soft clipped reads
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193 #############################################################
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194 #give file a name
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195 if(!defined($pair_only)){
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196 $random_file_sc=join ("",$random_name,".sc.sam");
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197 $command=join ("","samtools view -q $MapQ -F 1024 $INPUT_BAM $chrom $new_blacklist| awk '{OFS=\"\\t\"}{c=0;if(\$6~/S/){++c};if(c == 1){print}}' | perl -ane '\$TR=(\@F[10]=~tr/\#//);if(\$TR<2){print}' > ", $random_file_sc);
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198
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199 print "Making SAM file of soft-clipped reads\n";
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200 if($verbose){ print "$command\n";}
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201 system("$command");
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202
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203 #############################################################
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204 # Find areas that have deep enough soft-clip coverage
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205 print "Identifying soft-clipped regions that are at least $minSoft bp long \n";
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206 open (FILE,"$random_file_sc")||die "Can't open soft-clipped sam file $random_file_sc\n";
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207
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208 my $tmpfile=join("",$random_file_sc,".sc.passfilter");
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209 open (OUT,">$tmpfile")||die "Can't write files here!\n";
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210
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211 while(<FILE>){
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212 @_ = split(/\t/, $_);
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213 #### parse CIGAR string and create a hash of array of each operation
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214 my @CIGAR = split(/([0-9]+[SMIDNHXP])/, $_[5]);
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215 my $hash;
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216 map { push(@{$hash->{$2}}, $1) if (/(\d+)([SMIDNHXP])/) } @CIGAR;
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217
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218 #for ($i=0; $i<=$#softclip_pos; $i++) {
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219 foreach my $softclip (@{$hash->{S}}) {
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220 #if ($CIGAR[$softclip_pos[$i]] > $minSoft){
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221 if ($softclip > $minSoft){
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222 ###############Make sure base qualities don't have more than 2 bad marks
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223 my $qual=$_[10];
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224 my $TR=($qual=~tr/$badQualValue//);
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225 if($badQualValue eq "#"){ $TR=($qual=~tr/\#//); }
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226 #Skip the soft clip if there is more than 2 bad qual values
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227 #next if($TR > 2);
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228 # if (!$high_qual){next if($TR > 2);}
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229 print OUT;
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230 last;
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231 }
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232 }
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233 }
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234 close FILE;
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235 close OUT;
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236
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237 $command=join(" ","mv",$tmpfile,$random_file_sc);
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238 if($verbose){ print "$command\n";}
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239 system("$command");
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240 }
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241
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242 #########################################################
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243 #Stack up SoftClips
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244 #########################################################
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245 my $random_file=join("",$random_name,".sc.direction.bed");
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246 if(!defined($pair_only)){
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247 open (FILE,"$random_file_sc")|| die "Can't open sam file\n";
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248 #$random_file=join("",$random_name,".sc.direction");
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249
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250 print "Calling sides of soft-clips\n";
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251 #\nTMPOUT=$random_file\tINPUT=$random_file_sc\n\n";
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252 open (TMPOUT,">$random_file")|| die "Can't create tmp file\n";
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253
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254 while (<FILE>){
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255 @_ = split(/\t/, $_);
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256 #### parse CIGAR string and create a hash of array of each operation
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257 my @CIGAR = split(/([0-9]+[SMIDNHXP])/, $_[5]);
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258 my $hash;
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259 map { push(@{$hash->{$2}}, $1) if (/(\d+)([SMIDNHXP])/) } @CIGAR;
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260
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261 #### next if softclips on each end
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262 next if ($_[5] =~ /^[0-9]+S.*S$/);
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263
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264 #### next softclip occurs in the middle
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265 next if ($_[5] =~ /^[0-9]+[^S][0-9].*S.+$/);
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266
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267 my $softclip = $hash->{S}[0];
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268
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269 my $end1 = 0;
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270 my $end2 = 0;
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271 my $softBases = "";
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272 my $right_corrected="";my $left_corrected="";
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273
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274 if ($softclip > $minSoft) {
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275
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276 ####If the soft clip occurs at end of read and its on the minus strand, then it's a right clip
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277 if ($_[5] =~ /^.*S$/) {
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278 $end1=$_[3]+length($_[9])-$softclip-1;
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279 $end2=$end1+1;
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280 next if ($end1<0);
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281 #RIGHT clip on Minus
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282 $softBases=substr($_[9], length($_[9])-$softclip, length($_[9]));
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283 #Right clips don't always get clipped correctly, so fix that
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284 # Check to see if sc base matches ref
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285 $right_corrected=baseCheck($_[2],$end2,"right",$softBases);
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286 print TMPOUT "$right_corrected\n"
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287
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288 } else {
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289 #### Begins with S (left clip)
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290 $end1=$_[3]-$softclip;
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291 next if ($end1<0);
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292
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293 $softBases=substr($_[9], 0,$softclip);#print "TMP=$softBases\n";
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294 $left_corrected=baseCheck($_[2],$end1,"left",$softBases);
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295 if(!$left_corrected){print "baseCheck($_[2],$end1,left,$softBases)\n";next}
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296 print TMPOUT "$left_corrected\n";
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297 #print "\nSEQ=$_[9]\t\n";
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298
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299 }
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300 }
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301 }
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302 close FILE;
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303 close TMPOUT;
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304 }
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305 sub baseCheck{
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306 my ($chrom,$pos,$direction,$softBases)=@_;
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307 #skip if position is less than 0, which is caused by MT DNA
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308 return if ($pos<0);
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309 my $exit="";
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310
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311 while(!$exit){
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312 if($direction=~/right/){
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313 my $refBase=getSeq($chrom,$pos,$INPUT_FASTA);
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314 my $softBase=substr($softBases,0,1);
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315 if ($softBase !~ /$refBase/){
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316 my $value=join("\t",$chrom,$pos,$pos+1,join("|",$softBases,$direction));
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317 $exit="STOP";
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318 return $value;
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319 }
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320 else{
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321 $pos=$pos+1;
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322 $softBases=substr($softBases, 1,length($softBases));
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323 }
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324 }
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325 else{
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326 my $refBase=getSeq($chrom,$pos+1,$INPUT_FASTA);
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327 my $softBase=substr($softBases,-1,1);
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328 if ($softBase !~ /$refBase/){
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329 $pos=$pos-1+length($softBases);
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330 my $value=join("\t",$chrom,$pos-1,$pos,join("|",$softBases,$direction));
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331 $exit="STOP";
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332 return $value;
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333 }
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334 else{
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|
|
335 $pos=$pos-1;
|
|
|
336 $softBases=substr($softBases, 0, -1);
|
|
|
337 #print "Trying again $softBases\n";
|
|
|
338 }
|
|
|
339
|
|
|
340 }
|
|
|
341
|
|
|
342 }
|
|
|
343 }
|
|
|
344 #Remove SAM files to conserve space
|
|
|
345 unlink($random_file_sc);
|
|
|
346
|
|
|
347
|
|
|
348 my $random_file_disc="$INPUT_BAM";
|
|
|
349 ###
|
|
|
350 #
|
|
|
351 ######################################################
|
|
|
352 # Transform Read pair groups into softclip equivalents
|
|
|
353 ######################################################
|
|
|
354 #
|
|
|
355 #
|
|
|
356 #
|
|
|
357 my $v="";
|
|
|
358 #if($disable_RP_only){
|
|
|
359 print "Running Bam2pair.pl\n";
|
|
|
360 print "Looking for discordant read pairs without requiring soft-clipping information\n";
|
|
|
361 use FindBin qw($Bin);
|
|
|
362 my $path=$Bin;
|
|
|
363 # print"\n\nPATH=$path\n\n";
|
|
|
364 if($verbose){$v="-v"}
|
|
|
365 my $tmp_out=join("",$random_name,"RP.out");
|
|
|
366 $command=join("","perl ",$path,"/Bam2pair.pl -b $random_file_disc -o $tmp_out -isize $pe_upper_limit -winsize $dist_To_Soft -min $minRP -chrom $chrom -prefix $random_name -q $MapQ -blacklist $random_name.bed $v");
|
|
|
367 if($verbose){ print "$command\n"};
|
|
|
368 system("$command");
|
|
|
369 $command=join("","perl -ane '\$end1=\@F[1];\$end2=\@F[3];print join(\"\\t\",\@F[0..1],\$end1,\"unknown|left\");print \"\\n\";print join(\"\\t\",\@F[2..3],\$end2,\"unknown|left\");print \"\\n\"' ", $tmp_out," >> ",$random_file);
|
|
|
370 if($verbose){print "$command\n"};
|
|
|
371 system($command);
|
|
|
372 unlink($tmp_out);
|
|
|
373 #}
|
|
|
374 #
|
|
|
375
|
|
|
376
|
|
|
377 ######################################################
|
|
|
378 unlink("$random_file","$tmp_name","$random_file","$index","$random_name","$new_blacklist") if (-z $random_file || ! -e $random_file ) ;
|
|
|
379 if (-z $random_file || ! -e $random_file){
|
|
|
380 print "Softclipped file is empty($random_file).\nNo soft clipping found using desired paramters\n\n";
|
|
|
381 open (OUT,">$OUTNAME")||die "Can't write files here!\n";
|
|
|
382 &print_header();
|
|
|
383 close OUT;
|
|
|
384 exit;
|
|
|
385 }
|
|
|
386
|
|
|
387
|
|
|
388 #############################################################
|
|
|
389 # Make sure there are enough soft-clippped supporting reads
|
|
|
390 #############################################################
|
|
|
391 my $outfile=join("",$random_file,".sc.merge.bed");
|
|
|
392 #sortbed -i .sc.direction | mergeBed -nms -d 25 -i stdin > .sc.merge.bed
|
|
|
393 $command=join(" ","sortBed -i",$random_file," | mergeBed -nms -i stdin","|egrep \";|,\"","|awk '{OFS=\"\t\"}(NF==4)'",">",$outfile);
|
|
|
394
|
|
|
395 print "$command\n" if ($verbose);
|
|
|
396 system("$command");
|
|
|
397
|
|
|
398 if (-z $outfile || ! -e $outfile){
|
|
|
399 unlink("$tmp_name","$random_file","$outfile","$index","$random_name","$new_blacklist");
|
|
|
400 print "mergeBed file is empty.\nNo strucutral variants found\n\n" ;
|
|
|
401 open (OUT,">$OUTNAME")||die "Can't write files here!\n";
|
|
|
402 &print_header();
|
|
|
403 close OUT;
|
|
|
404 exit;
|
|
|
405 }
|
|
|
406
|
|
|
407 print "completed mergeBed\n";
|
|
|
408
|
|
|
409 ###############################################################
|
|
|
410 # If left and right are on the same line, make into 2 lines
|
|
|
411 ###############################################################
|
|
|
412 open (INFILE,$outfile)||die "couldn't open temp file : $. \n\n";
|
|
|
413 my $tmp2=join("",$random_name,".sc.fixed.merge.bed");
|
|
|
414 #print "INFILE=$outfile\tOUTFILE=$tmp2\n\n";
|
|
|
415 #INPUT FORMAT=chr9\t131467\t131473\tATGCTTATTAAAA|left;TTATTAAAAGCATA|left
|
|
|
416 open (OUTFILE,">$tmp2")||die "couldn't create temp file : $. \n\n";
|
|
|
417 while(<INFILE>){
|
|
|
418 chomp $_;
|
|
|
419 my $l = $_;
|
|
|
420
|
|
|
421 my @a = split(/\t/, $l);
|
|
|
422 my $info = $a[3];
|
|
|
423 my @info_arr = split(/\;/, $info);
|
|
|
424 my @left_arr=();
|
|
|
425 my @right_arr=();
|
|
|
426 @left_arr = grep(/left/, @info_arr);
|
|
|
427 @right_arr = grep(/right/, @info_arr);
|
|
|
428
|
|
|
429 #New
|
|
|
430 my $left = join(";", @left_arr);
|
|
|
431 my $right = join(";", @right_arr);
|
|
|
432 $info = join(";", @info_arr);
|
|
|
433
|
|
|
434 if((@left_arr) && (@right_arr)){
|
|
|
435 print OUTFILE "$a[0]\t$a[1]\t$a[2]\t$left\n$a[0]\t$a[1]\t$a[2]\t$right\n";
|
|
|
436 } else{
|
|
|
437 my $all=join("\t",@a[0..2],$info);
|
|
|
438 print OUTFILE "$all\n";
|
|
|
439 }
|
|
|
440 }
|
|
|
441
|
|
|
442 # make sure output file name is $outfile
|
|
|
443 $command=join(" ","sed -e '/ /s//\t/g'", $tmp2,"|awk 'BEGIN{OFS=\"\\t\"}(NF==4)'", "|perl -pne 's/ /\t/g'>",$outfile);
|
|
|
444 system("$command");
|
|
|
445 if($verbose){print "$command\n"};
|
|
|
446 unlink("$tmp_name","$random_file","$tmp2","$outfile","$index","random_name","$new_blacklist") if (-z $outfile || ! -e $outfile) ;
|
|
|
447 if (-z $outfile || ! -e $outfile){
|
|
|
448 print "Fixed mergeBed file is empty($outfile).\nNo strucutral variants found\n\n";
|
|
|
449 open (OUT,">$OUTNAME")||die "Can't write files here!\n";
|
|
|
450 &print_header();
|
|
|
451 close OUT;
|
|
|
452 exit;
|
|
|
453 }
|
|
|
454
|
|
|
455 print "completed fixing mergeBed\n\n";
|
|
|
456
|
|
|
457 ###############################################################
|
|
|
458 # Seperate directions of soft clips
|
|
|
459 ###############################################################
|
|
|
460 my $left_sc = join("", "left", $tmp2);
|
|
|
461 my $right_sc = join("", "right", $tmp2);
|
|
|
462 use FindBin qw($Bin);
|
|
|
463 #my $path=$Bin;
|
|
|
464
|
|
|
465 $command=join("","grep left ", $tmp2, " |sed -e '/left /s//left\;/g' | sed -e '/ /s//\t/g'|perl ".$path."/direction_filter.pl - >",$left_sc);
|
|
|
466 system("$command");
|
|
|
467 #print "$command\n";
|
|
|
468 $command=join("","grep right ", $tmp2, " |sed -e '/right /s//right\;/g' | sed -e '/ /s//\t/g'|perl ".$path."/direction_filter.pl - >",$right_sc);
|
|
|
469 #$command=join(" ","grep right ", $tmp2, " |sed -e '/right /s//right\;/g' | sed -e '/ /s//\t/g' >",$right_sc);
|
|
|
470 system("$command");
|
|
|
471 #print "$command\n";
|
|
|
472 #die "CHECK $right_sc\n";
|
|
|
473
|
|
|
474 ###############################################################
|
|
|
475 # Count the number and identify directions of soft clips
|
|
|
476 ###############################################################
|
|
|
477 print "Count the number and identify directions of soft clips\n";
|
|
|
478 #print "looking in $outfile\n";
|
|
|
479 $outfile=join("",$random_name,".sc.fixed.merge.bed");
|
|
|
480
|
|
|
481 open (INFILE,$outfile)||die "couldn't open temp file\n\n";
|
|
|
482 my $tmp3 = join("", $random_file, "predSV");
|
|
|
483 open (OUTFILE, ">$tmp3")||die "couldn't create temp file\n\n";
|
|
|
484 while(<INFILE>){
|
|
|
485 chomp;
|
|
|
486 @_=split(/\t/,$_);
|
|
|
487 my $count=tr/\;//;$count+=tr/\,//;
|
|
|
488 $count=$count+1;
|
|
|
489 my $left=0;
|
|
|
490 my $right=0;
|
|
|
491
|
|
|
492 while ($_ =~ /left/g) { $left++ } # count number of right clips
|
|
|
493 while ($_ =~ /right/g) { $right++ } # count number of left clips
|
|
|
494
|
|
|
495 ###############################################################
|
|
|
496 if ($count >= $minSoftReads){
|
|
|
497 ####get longets soft-clipped read
|
|
|
498 my @clips=split(/\;|,|\|/,$_[3]);
|
|
|
499
|
|
|
500 my ($max, $temp, $temp2, $temp3, $dir, $maxSclip) = (0) x 6;
|
|
|
501 for (my $i=0; $i<$count; $i++) {
|
|
|
502 my $plus1=$i+1;
|
|
|
503 $temp=length($clips[$i]);
|
|
|
504 $temp2=$clips[$plus1];
|
|
|
505 $temp3=$clips[$i];
|
|
|
506
|
|
|
507 if ($temp > $max){
|
|
|
508 $maxSclip=$temp3;
|
|
|
509 $max =$temp;
|
|
|
510 $dir=$temp2;
|
|
|
511 } else {
|
|
|
512 $max=$max;
|
|
|
513 $dir=$dir;
|
|
|
514 $maxSclip=$maxSclip;
|
|
|
515 }
|
|
|
516 $i++;
|
|
|
517 }
|
|
|
518 my $order2 = join("|", $left, $right);
|
|
|
519 #print join ("\t",@_[0..2],$maxSclip,$max,$dir,$count,$order2) . "\n";
|
|
|
520 print OUTFILE join ("\t",@_[0..2],$maxSclip,$max,$dir,$count,$order2) . "\n";
|
|
|
521 } elsif($_=~/unknown/){
|
|
|
522 print OUTFILE join ("\t",@_[0..2],"NA","NA","left","NA","NA|NA") . "\n";
|
|
|
523 print OUTFILE join ("\t",@_[0..2],"NA","NA","right","NA","NA|NA") . "\n";
|
|
|
524 }
|
|
|
525 ####Format is Chrom,start, end,longest Soft-clip,length of longest Soft-clip, direction of longest soft-clip,#supporting softclips,#right Sclips|#left Sclips
|
|
|
526 }
|
|
|
527 close INFILE;
|
|
|
528 close OUTFILE;
|
|
|
529
|
|
|
530 unlink("$tmp2","$tmp_name","$random_file","$tmp3","$outfile","$index","$random_name","$right_sc","$left_sc","$new_blacklist") if (-z $tmp3 || !-e $tmp3) ;
|
|
|
531
|
|
|
532 if (-z $tmp3 || !-e $tmp3){
|
|
|
533 print "No structural variants found while Counting the number and identify directions of soft clips.\n" ;
|
|
|
534
|
|
|
535 open (OUT,">$OUTNAME")||die "Can't write files here!\n";
|
|
|
536 &print_header();
|
|
|
537 close OUT;
|
|
|
538 exit;
|
|
|
539
|
|
|
540 }
|
|
|
541
|
|
|
542 print "Done counting Softclipped reads\n";
|
|
|
543 ###############################################################
|
|
|
544 #### Print header information
|
|
|
545 ###############################################################
|
|
|
546 open (OUT,">$OUTNAME")||die "Can't write files here!\n";
|
|
|
547 &print_header();
|
|
|
548 close OUT;
|
|
|
549
|
|
|
550 ###############################################################
|
|
|
551 ###############################################################
|
|
|
552 #### DO the bulk of the work
|
|
|
553 ###############################################################
|
|
|
554 use List::Util qw(min max);
|
|
|
555 open (FILE,"$tmp3")|| die "Can't open file\n";
|
|
|
556 open (OUT,">>$OUTNAME")|| die "Can't open file\n";
|
|
|
557
|
|
|
558 #print "\nusing $tmp3 and writing to $OUTPUT_FILE \n";
|
|
|
559 while (<FILE>){
|
|
|
560 #If left clip {+- or -- or -+ }{+- are uninformative b/c they go upstream}
|
|
|
561 #If right clip {++ or -- or +-}
|
|
|
562 chomp $_;
|
|
|
563 my $line = $_;
|
|
|
564 my @info = split(/\t/, $_);
|
|
|
565
|
|
|
566 if($info[5] eq "left") {
|
|
|
567 bulk_work("left", $line, $random_file_disc);
|
|
|
568
|
|
|
569 } elsif ($info[5] eq "right") {
|
|
|
570 bulk_work("right", $line, $random_file_disc);
|
|
|
571 }
|
|
|
572 #if($. ==6){print "THIS IS LINE 6\n$_\n";die}
|
|
|
573 print "Completed line $.\n" if ($verbose);
|
|
|
574 }
|
|
|
575 close FILE;
|
|
|
576 close OUT;
|
|
|
577
|
|
|
578 ###############################################################################
|
|
|
579 ###############################################################################
|
|
|
580 #### Delete temp files
|
|
|
581 my $meregedBed=join("",$random_name,".sc.direction.bed.sc.merge.bed");
|
|
|
582
|
|
|
583 if(defined($temp_output)){$temp_output=$temp_output} else {$temp_output="no"}
|
|
|
584
|
|
|
585 if ($temp_output eq "no"){
|
|
|
586 unlink("$tmp_name","$random_file","$tmp2",,"$tmp3","$outfile","$index","$random_name","$right_sc","$left_sc","$meregedBed","$random_name.bed");
|
|
|
587 }
|
|
|
588 ####Sort VCF
|
|
|
589 my $tmp=join(".",$random_name,"tmp");
|
|
|
590 #Get header
|
|
|
591 $cmd="grep \"#\" $OUTNAME > $tmp";
|
|
|
592 system($cmd);
|
|
|
593 #sort results
|
|
|
594 $cmd="grep -v \"#\" $OUTNAME|perl -pne 's/chr//'|sort -k1,1n -k2,2n|perl -ne 'print \"chr\".\$_' >>$tmp";
|
|
|
595 system($cmd);
|
|
|
596 $cmd="mv $tmp $OUTNAME";
|
|
|
597 system($cmd);
|
|
|
598 #remove entries next to each other
|
|
|
599
|
|
|
600
|
|
|
601
|
|
|
602
|
|
|
603 #############################################################
|
|
|
604 ##May not need this anymore since filtering on left and right
|
|
|
605 #############################################################
|
|
|
606 #my $tmpout=$OUTNAME;
|
|
|
607 #$tmpout.=".tmp";
|
|
|
608 #use FindBin qw($Bin);
|
|
|
609 ##my $path=$Bin;
|
|
|
610 #$command="perl ".$path."/Extract_nSC.pl $OUTNAME -q nSC > $tmpout";
|
|
|
611 ##print "Command=$command\n";
|
|
|
612 #system($command);
|
|
|
613 #$command="perl ".$path."/reduce_redundancy.pl $tmpout $upper_limit |cut -f1-10 > $OUTNAME";
|
|
|
614 ##print "$command\n";
|
|
|
615 #system($command);
|
|
|
616 #system("rm $tmpout");
|
|
|
617 ########################################################
|
|
|
618
|
|
|
619
|
|
|
620
|
|
|
621
|
|
|
622 print "Analysis Completed\n\nYou did it!!!\n";
|
|
|
623 print "Finish Time : " . &spGetCurDateTime() . "\n";
|
|
|
624 $now = time - $now;
|
|
|
625 printf("\n\nTotal running time: %02d:%02d:%02d\n\n", int($now / 3600), int(($now % 3600) / 60),
|
|
|
626 int($now % 60));
|
|
|
627
|
|
|
628 exit;
|
|
|
629
|
|
|
630 ###############################################################################
|
|
|
631 sub rev_comp {
|
|
|
632 my $dna = shift;
|
|
|
633 my $revcomp = reverse($dna);
|
|
|
634 $revcomp =~ tr/ACGTacgt/TGCAtgca/;
|
|
|
635
|
|
|
636 return $revcomp;
|
|
|
637 }
|
|
|
638
|
|
|
639
|
|
|
640 ###############################################################################
|
|
|
641 #### to get reference base
|
|
|
642 sub getSeq{
|
|
|
643 my ($chr,$pos,$fasta)=@_;
|
|
|
644 #don't require chr
|
|
|
645 #if($chr !~ /^chr/){die "$chr is not correct\n";}
|
|
|
646 # die "$pos is not a number\n" if ($pos <0);
|
|
|
647 my @result=();
|
|
|
648 if ($pos <0){print "$pos is not a valid position (likely caused by circular MT chromosome)\n";return;}
|
|
|
649
|
|
|
650 @result = `samtools faidx $fasta $chr:$pos-$pos`;
|
|
|
651 if($result[1]){chomp($result[1]);
|
|
|
652 return uc($result[1]);
|
|
|
653 }
|
|
|
654 return("NA");
|
|
|
655 #### after return will not be printed
|
|
|
656 ####print "RESULTS=@result\n";
|
|
|
657 }
|
|
|
658
|
|
|
659 sub getBases{
|
|
|
660 my ($chr,$pos1,$pos2,$fasta)=@_;
|
|
|
661 #don't require chr
|
|
|
662 #if($chr !~ /^chr/){die "$chr is not correct\n";}
|
|
|
663 my @result=();
|
|
|
664 if ($pos1 <0){print "$pos1 is not a valid position (likely caused by circular MT chromosome)\n";return;};
|
|
|
665
|
|
|
666 @result = `samtools faidx $fasta $chr:$pos1-$pos2`;
|
|
|
667 if(!$result[1]){$result[1]="NA"};
|
|
|
668 chomp($result[1]);
|
|
|
669 return uc($result[1]);
|
|
|
670
|
|
|
671 #### after return will not be printed
|
|
|
672 ####print "RESULTS=@result\n";
|
|
|
673 }
|
|
|
674 ###############################################################################
|
|
|
675 #### to get time
|
|
|
676 sub spGetCurDateTime {
|
|
|
677 my ($sec, $min, $hour, $mday, $mon, $year) = localtime();
|
|
|
678 my $curDateTime = sprintf "%4d-%02d-%02d %02d:%02d:%02d",
|
|
|
679 $year+1900, $mon+1, $mday, $hour, $min, $sec;
|
|
|
680 return ($curDateTime);
|
|
|
681 }
|
|
|
682
|
|
|
683
|
|
|
684 ###############################################################################
|
|
|
685 #### print header
|
|
|
686 sub print_header {
|
|
|
687 my $date=&spGetCurDateTime();
|
|
|
688 my $header = qq{##fileformat=VCFv4.1
|
|
|
689 ##fileDate=$date
|
|
|
690 ##source=SoftSearch.pl
|
|
|
691 ##reference=$INPUT_FASTA
|
|
|
692 ##Usage= SoftSearch.pl -l $minSoft -q $MapQ -r $minSoftReads -d $dist_To_Soft -m $minRP -u $unmated_pairs -s $num_sd -b $INPUT_BAM -f $INPUT_FASTA -o $OUTNAME
|
|
|
693 ##INFO=<ID=SVTYPE,Number=1,Type=String,Description="Type of structural variant">
|
|
|
694 ##INFO=<ID=EVENT,Number=1,Type=String,Description="ID of event associated to breakend">
|
|
|
695 ##INFO=<ID=MATEID,Number=.,Type=String,Description="ID of mate breakends">
|
|
|
696 ##INFO=<ID=ISIZE,Number=.,Type=String,Description="Size of the SV">
|
|
|
697 ##INFO=<ID=END,Number=1,Type=Integer,Description="End position of the variant described in this record">
|
|
|
698 ##FORMAT=<ID=lSC,Number=1,Type=Integer,Description="Length of the longest soft clips supporting the BND">
|
|
|
699 ##FORMAT=<ID=nSC,Number=1,Type=Integer,Description="Number of supporting soft-clips\">
|
|
|
700 ##FORMAT=<ID=uRP,Number=1,Type=Integer,Description="Number of unmated read pairs nearby Soft-Clips">
|
|
|
701 ##FORMAT=<ID=levD_local,Number=1,Type=Float,Description="Levenstein distance between soft-clipped bases and the area around the original soft-clipped site">
|
|
|
702 ##FORMAT=<ID=levD_distl,Number=1,Type=Float,Description="Levenstein distance between the soft-clipped bases and mate location">
|
|
|
703 ##FORMAT=<ID=CTX,Number=1,Type=Integer,Description="Number of chromosomal translocations">
|
|
|
704 ##FORMAT=<ID=DEL,Number=1,Type=Integer,Description="Number of reads supporting Large Deletions">
|
|
|
705 ##FORMAT=<ID=INS,Number=1,Type=Integer,Description="Number of reads supporting Large insertions">
|
|
|
706 ##FORMAT=<ID=NOV_INS,Number=1,Type=Integer,Description="Number of reads supporting novel sequence insertion">
|
|
|
707 ##FORMAT=<ID=TDUP,Number=1,Type=Integer,Description="Number of reads supporting a tandem duplication">
|
|
|
708 ##FORMAT=<ID=INV,Number=1,Type=Integer,Description="Number of reads supporting inversions">
|
|
|
709 ##FORMAT=<ID=sDEL,Number=1,Type=Integer,Description="Number of reads supporting novel sequence insertion">
|
|
|
710 ##INFO=<ID=NO_MATE_SC,Number=1,Type=Flag,Description="When there is no softclipping of the mate read location, an appromiate position is used">
|
|
|
711 ##FORMAT=<ID=GT,Number=1,Type=String,Description="Dummy value for maintaining VCF-Spec">
|
|
|
712 #CHROM\tPOS\tID\tREF\tALT\tQUAL\tFILTER\tINFO\tFORMAT\t$SAMPLE_NAME\n};
|
|
|
713
|
|
|
714 print OUT $header;
|
|
|
715 }
|
|
|
716
|
|
|
717
|
|
|
718 ###############################################################################
|
|
|
719 sub bulk_work {
|
|
|
720 print "#####################################@_\n" if ($verbose);
|
|
|
721 my ($side, $line, $file) = @_;
|
|
|
722 my $local_levD = 0;
|
|
|
723 my $distl_levD = 0;
|
|
|
724
|
|
|
725 #my @info = split(/\t/, $line);
|
|
|
726 my @plus_Reads = split(/\t/, $line);
|
|
|
727 $plus_Reads[7] =~ s/\n//g;
|
|
|
728
|
|
|
729 #### softclip length and softclip size.
|
|
|
730 my $lSC = $plus_Reads[4];
|
|
|
731 my $nSC = $plus_Reads[6];
|
|
|
732
|
|
|
733
|
|
|
734 #Get all types of compatible reads
|
|
|
735 #Get improperly paired reads (@ max distance)
|
|
|
736
|
|
|
737 #### default value for left SIDE.
|
|
|
738 #If left-clip, then look downstream for match of softclipped reads to define a deletion, but look for DRPs upstream
|
|
|
739 my $sv_type = "SVTYPE=BND";
|
|
|
740 my $start_local=0; my $end_local=0;my $target_local="";my $target_drp="";my $start_drp="";my $end_drp="";
|
|
|
741 if ($side =~ /left/) {
|
|
|
742 $start_local = $plus_Reads[1]-$dist_To_Soft;
|
|
|
743 $end_local = $plus_Reads[2];
|
|
|
744 $start_drp = $plus_Reads[1];
|
|
|
745 $end_drp = $plus_Reads[1]+$dist_To_Soft;
|
|
|
746
|
|
|
747 }
|
|
|
748 else{
|
|
|
749 $start_local = $plus_Reads[1];
|
|
|
750 $end_local = $plus_Reads[1]+$dist_To_Soft;
|
|
|
751 $start_drp = $plus_Reads[1]-$dist_To_Soft;
|
|
|
752 $end_drp = $plus_Reads[1];
|
|
|
753 }
|
|
|
754
|
|
|
755 $target_local=join("", $plus_Reads[0], ":", $start_local, "-", $end_local);
|
|
|
756 $target_drp=join("", $plus_Reads[0], ":", $start_drp, "-", $end_drp);
|
|
|
757 my $num_unmapped_pairs="";
|
|
|
758 if ($side =~ /right/) {
|
|
|
759 $num_unmapped_pairs=`samtools view $new_blacklist -q $MapQ -f8 -F 1536 -c $INPUT_BAM $target_drp`;
|
|
|
760 } else {
|
|
|
761 $num_unmapped_pairs=`samtools view $new_blacklist -q $MapQ -f24 -F 1536 -c $INPUT_BAM $target_drp`;
|
|
|
762 }
|
|
|
763 if($verbose){print "samtools view $new_blacklist -q $MapQ -f24 -F 1536 -c $INPUT_BAM $target_drp\n";}
|
|
|
764
|
|
|
765 $num_unmapped_pairs=~s/\n//;
|
|
|
766 if($verbose){print "NUM UNMAPPED PAIRS= $num_unmapped_pairs\n";}
|
|
|
767 my $REF1_base = "";
|
|
|
768 my $REF2_base = "";
|
|
|
769 my $INFO_1 = "";
|
|
|
770 my $INFO_2 = "";
|
|
|
771 my $ALT_1 = "";
|
|
|
772 my $ALT_2 = "";
|
|
|
773 my $isize = 0;
|
|
|
774 my $QUAL = "";
|
|
|
775 my $FORMAT = "GT:";
|
|
|
776
|
|
|
777 #### get 8 bit rand id
|
|
|
778 my $BND1_name = join "", map { ("a".."z")[rand 26] } 1..8;
|
|
|
779 my $BND2_name = join "", map { ("a".."z")[rand 26] } 1..8;
|
|
|
780 $BND1_name=join "_","BND",$BND1_name;
|
|
|
781 $BND2_name=join "_","BND",$BND2_name;
|
|
|
782
|
|
|
783 my $counts = {CTX => 0, DEL => 0, INS => 0, INV => 0, TDUP => 0, NOV_INS => 0 };
|
|
|
784 my $event_mate_info = {CTX => "", DEL => "", INS => "", INV => "", TDUP => "", NOV_INS => "" };
|
|
|
785
|
|
|
786 #### get mate pair info and counts per event
|
|
|
787 foreach my $e (sort keys %{$counts}) {
|
|
|
788 my $h = get_counts_n_info($e, $side, $MapQ, $file, $dist_To_Soft, $target_drp, $upper_limit, $lower_limit);
|
|
|
789
|
|
|
790 $counts->{$e} = $h->{count};
|
|
|
791 $event_mate_info->{$e} = $h->{info};
|
|
|
792 }
|
|
|
793 #print Dumper($counts);
|
|
|
794
|
|
|
795 my $max = 0;
|
|
|
796 my $type = "UNKNOWN";
|
|
|
797 my $nRP = 0;
|
|
|
798 my $mate_info = "NA\tNA\tNA\tNA";
|
|
|
799 my $summary = "GT:";
|
|
|
800
|
|
|
801 #### find max count of events and set type, nRP and info to corresponding
|
|
|
802 #### max count event.
|
|
|
803 #### also create a summary string of all counts to be added to VCF file.
|
|
|
804 foreach my $e (sort keys %{$counts}){
|
|
|
805 # if ($counts->{$e} >=i $max){
|
|
|
806 if ($counts->{$e} > $max){
|
|
|
807 $type = $e .",". $counts->{$e};
|
|
|
808 $nRP = $counts->{$e};
|
|
|
809
|
|
|
810 $max = $counts->{$e};
|
|
|
811
|
|
|
812 if (length($event_mate_info->{$e})) {
|
|
|
813 $mate_info = $event_mate_info->{$e};
|
|
|
814 }
|
|
|
815 }
|
|
|
816
|
|
|
817 $summary .= $e .",". $counts->{$e} .":";
|
|
|
818 }
|
|
|
819 # print "done with Summary\n";
|
|
|
820 #### remove last colon ":" from
|
|
|
821 $summary =~ s/:$//;
|
|
|
822 if (($minRP > $max)&&(!$disable_RP_only )){if ($verbose){print "FAILED BECAUSE ($minRP > $max)&&(!$disable_RP_only )"};return};
|
|
|
823
|
|
|
824 #### Run Levenstein distance on softclip in target region to find out if its a small deletion/insetion
|
|
|
825 #### passing 1: clip_seq, 2: chr, 3: start, 4: end, 5: ref file.
|
|
|
826 my $levD = new LevD;
|
|
|
827 ########################################################
|
|
|
828 ########################################################
|
|
|
829 ########################################################
|
|
|
830
|
|
|
831 #### redefine start and end location for LevD calc.
|
|
|
832 # $start = $plus_Reads[1]-$dist_To_Soft;
|
|
|
833 # $end = $plus_Reads[2];
|
|
|
834 my $num_bases_to_loc=0;
|
|
|
835 my $new_start=0;
|
|
|
836 my $new_end=0;
|
|
|
837 my $del_seq="";
|
|
|
838 my $start = $start_local;
|
|
|
839 my $end = $end_local;
|
|
|
840 if ($lSC=~/NA/){$lSC=0}
|
|
|
841
|
|
|
842 if ($side =~ /right/) {
|
|
|
843 $levD->search($plus_Reads[3], $plus_Reads[0], $start, $end, $INPUT_FASTA);
|
|
|
844 $local_levD = sprintf("%.2f", $levD->{relative_edit_dist});
|
|
|
845 $num_bases_to_loc=$levD->{index};
|
|
|
846 $new_start = $plus_Reads[2];
|
|
|
847 if ($plus_Reads[2]=~/^[0-9]/){$new_end=$plus_Reads[2]+$lSC};
|
|
|
848 }
|
|
|
849 else{
|
|
|
850 $levD->search($plus_Reads[3], $plus_Reads[0], $start, $end, $INPUT_FASTA);
|
|
|
851 $local_levD = sprintf("%.2f", $levD->{relative_edit_dist});
|
|
|
852 $num_bases_to_loc=$levD->{index};
|
|
|
853 if ($plus_Reads[2]=~/^[0-9]/){$new_start=$plus_Reads[2]-$lSC};
|
|
|
854 $new_end = $plus_Reads[2];
|
|
|
855 }
|
|
|
856 if((!$new_start)||(!$new_end)||($new_start<0)){print "FAILED AT ((!$new_start)||(!$new_end)||($new_start<0))\n";return};
|
|
|
857
|
|
|
858 $del_seq=getBases($plus_Reads[0], $new_start,$new_end,$INPUT_FASTA);
|
|
|
859 ##############################################################################
|
|
|
860 # #If there is a match, where is the start position of the match?
|
|
|
861 #
|
|
|
862 ##############################################################################
|
|
|
863
|
|
|
864
|
|
|
865 #if $plus_Reads[3] eq "NA", then it was found without soft-clipped reads
|
|
|
866 if($plus_Reads[3] !~ /NA/){
|
|
|
867 if (($local_levD < $levD_local_threshold)) {
|
|
|
868 return if (!$sv_only);
|
|
|
869 #### add value to summary to be written to vcf file.
|
|
|
870 $summary = "GT:sDel," . $plus_Reads[6];
|
|
|
871 $type = "sDEL";
|
|
|
872 ###########################################################################
|
|
|
873 ##### Printing output
|
|
|
874
|
|
|
875 #########################################
|
|
|
876 ##### Get DNA info
|
|
|
877 #########################################
|
|
|
878 #$REF1_base = getSeq($plus_Reads[0], $plus_Reads[1], $INPUT_FASTA);
|
|
|
879 $REF1_base = substr($del_seq, 0, 1);
|
|
|
880
|
|
|
881 #### this is alt ref. for softclip its $plus_Reads[3]
|
|
|
882 $REF2_base = $del_seq;
|
|
|
883 $QUAL = 1/($local_levD + 0.001);
|
|
|
884 $QUAL = sprintf("%.2f",$QUAL);
|
|
|
885 $isize = length($del_seq);
|
|
|
886
|
|
|
887 #### svtype = none for sDEL
|
|
|
888 #### isize = length($info[3]);
|
|
|
889 #### nRP = NA
|
|
|
890 #### mate_id = NA
|
|
|
891 #### CTX,:DEL,:....sDEL,##
|
|
|
892 $INFO_1=join(";", "SVTYPE=NA", "EVENT=$type", "ISIZE=$isize");
|
|
|
893
|
|
|
894 #Add Sample infomration
|
|
|
895 my $FORMAT="GT:sDEL";
|
|
|
896 $FORMAT .= ":lSC:nSC:uRP:levD_local";
|
|
|
897 my $SAMPLE= "0/1:";
|
|
|
898 $SAMPLE .= "$plus_Reads[6]:$lSC:$nSC:$num_unmapped_pairs:$local_levD";
|
|
|
899
|
|
|
900 #### remove any white spaces.
|
|
|
901 $INFO_1=~s/\s//g;
|
|
|
902 $INFO_2=~s/\s//g;
|
|
|
903
|
|
|
904 $BND1_name =~ s/^BND/LEVD/;
|
|
|
905 # If left, then the start position is plus_Reads[1]-isize
|
|
|
906 my $start_pos=0;
|
|
|
907 #Make sure Ref1 and Ref2 bases are different
|
|
|
908 if($REF2_base eq $REF1_base){$REF1_base="NA"}
|
|
|
909 if($side=~/left/){$start_pos=$plus_Reads[1]-$isize}else{$start_pos=$plus_Reads[1]};
|
|
|
910 print OUT join ("\t", $plus_Reads[0], $start_pos, $BND1_name, $REF2_base, $REF1_base, $QUAL, "PASS", $INFO_1,$FORMAT,$SAMPLE, "\n");
|
|
|
911 if ($verbose){print "No Softclipped reads here!\n"}
|
|
|
912 return;
|
|
|
913 }
|
|
|
914 }
|
|
|
915
|
|
|
916 #### Otherwise, look for DRP mate info
|
|
|
917 #if($nRP=~/NA/){print "MATE_INFO=$mate_info\tSide=$side\tline=$line\n";}
|
|
|
918 my @mate_info_arr = split(/\t/, $mate_info);
|
|
|
919 $nRP = $mate_info_arr[3];
|
|
|
920 my $mate_chr=$mate_info_arr[0];
|
|
|
921
|
|
|
922 if((! defined $nRP) || ($nRP =~ /na/i) || ($mate_chr =~ /NA/) ){
|
|
|
923 #PRINT UNKNOWN
|
|
|
924 if ($nRP =~ /na/i){print "Can't find SC reads\n" if ($verbose);return};
|
|
|
925 if ($verbose){print "There is an unknown\nNRP=$nRP Mate_CHR=$mate_chr minRP=$minRP\n"}
|
|
|
926 $summary .= ":unknown," . $plus_Reads[6];
|
|
|
927 $type = "unknown";
|
|
|
928 $REF1_base = getSeq($plus_Reads[0], $plus_Reads[1], $INPUT_FASTA);
|
|
|
929 $REF2_base = $plus_Reads[3];
|
|
|
930 $BND1_name =~ s/^BND/UNKNOWN/;
|
|
|
931 $QUAL = 1/($local_levD + 0.001);
|
|
|
932 $QUAL = sprintf("%.2f",$QUAL);
|
|
|
933 $INFO_1=join(";", "SVTYPE=unknown", "EVENT=unknown", "ISIZE=unknown");
|
|
|
934 #Add Sample infomration
|
|
|
935 my $FORMAT="GT:sDEL";
|
|
|
936 $FORMAT .= ":lSC:nSC:uRP:levD_local";
|
|
|
937 my $SAMPLE = "0/1:";
|
|
|
938 $SAMPLE .= "$plus_Reads[6]:$lSC:$nSC:$num_unmapped_pairs:$local_levD";
|
|
|
939 $SAMPLE=~s/NA/0/g;
|
|
|
940 #### remove any white spaces.
|
|
|
941 $INFO_1=~s/\s//g;
|
|
|
942 #print join ("\t", $plus_Reads[0], $plus_Reads[1], $REF2_base, $REF1_base, $QUAL, "PASS", $INFO_1,$FORMAT,$SAMPLE, "\n");
|
|
|
943
|
|
|
944 print OUT join ("\t", $plus_Reads[0], $plus_Reads[1], $BND1_name, $REF1_base, $REF2_base, $QUAL, "PASS", $INFO_1,$FORMAT,$SAMPLE, "\n");
|
|
|
945 return;
|
|
|
946
|
|
|
947 }
|
|
|
948 #### end if there is no mate info or nRP+uRP<minRP
|
|
|
949 if (($nRP<$minRP)&&($unmated_pairs > ($num_unmapped_pairs+$nRP))){
|
|
|
950 print "Something failed here\nif (($nRP<$minRP)&&($unmated_pairs > ($num_unmapped_pairs+$nRP)))\n";
|
|
|
951 return}
|
|
|
952
|
|
|
953 ##################################################################################
|
|
|
954 # Find out if mates have nearby soft-clips (to refine the breakpoints)
|
|
|
955 ##################################################################################
|
|
|
956 #Look for evidence of soft-clipping near mate
|
|
|
957 my @mate_soft_arr = ();
|
|
|
958 my $mate_start = 0;
|
|
|
959 my $mate_soft = "";
|
|
|
960
|
|
|
961 @mate_info_arr = split(/\t/, $mate_info);
|
|
|
962
|
|
|
963 #### mate start and end locations.
|
|
|
964 my $filename = $right_sc;
|
|
|
965
|
|
|
966 $start = $mate_info_arr[1] - $dist_To_Soft;
|
|
|
967 $end = $mate_info_arr[1];
|
|
|
968
|
|
|
969 if ($side =~ /right/) {
|
|
|
970 $start = $mate_info_arr[2];
|
|
|
971 $end = $mate_info_arr[2] + $dist_To_Soft;
|
|
|
972
|
|
|
973 $filename = $left_sc;
|
|
|
974 }
|
|
|
975
|
|
|
976 #### add levenstein distance to Summary
|
|
|
977 #print "Calc distal Levd\n";
|
|
|
978 $levD->search(rev_comp($plus_Reads[3]), $mate_info_arr[0], $start, $end, $INPUT_FASTA);
|
|
|
979 $distl_levD = sprintf("%.2f", $levD->{relative_edit_dist});
|
|
|
980 $distl_levD = "NA" if($plus_Reads[3] =~ /NA/);
|
|
|
981 #If there is no softclips to string match, then give 0 as quality value
|
|
|
982 if ($plus_Reads[3] !~ /NA/){
|
|
|
983 $QUAL=1/($distl_levD + 0.001);
|
|
|
984 }
|
|
|
985 else {
|
|
|
986 $QUAL=0;
|
|
|
987 };
|
|
|
988 $QUAL=sprintf("%.2f",$QUAL);
|
|
|
989 #### looking for softclips to refine break point
|
|
|
990 #### if left look in right and vice-versa.
|
|
|
991 $cmd = qq{echo -e "$mate_info_arr[0]\t$start\t$end"};
|
|
|
992 $cmd .= qq{ | awk -F'\t' 'NF==3' | intersectBed -a stdin -b $filename | head -1};
|
|
|
993 print "$cmd\n" if $verbose;
|
|
|
994 $mate_soft = `$cmd`;
|
|
|
995
|
|
|
996 $mate_soft =~ s/\n//g;
|
|
|
997 @mate_soft_arr = split(/\s/, $mate_soft);
|
|
|
998 my $NO_MATE_SC="";
|
|
|
999 if(@mate_soft_arr){
|
|
|
1000 $mate_chr = $mate_soft_arr[0];
|
|
|
1001 $mate_start = $mate_soft_arr[1];
|
|
|
1002 $NO_MATE_SC="APPROXIMATE";
|
|
|
1003
|
|
|
1004 } else{
|
|
|
1005 @mate_info_arr = split(/\s/,$mate_info);
|
|
|
1006 $mate_chr = $mate_info_arr[0];
|
|
|
1007 $mate_start = $mate_info_arr[1];
|
|
|
1008 }
|
|
|
1009
|
|
|
1010 #end if there is no mate info
|
|
|
1011 return if ($mate_chr eq "");
|
|
|
1012 #end if there is no mate info and !disable_RP_only
|
|
|
1013 return if (($lSC =~/NA/)&&(!$disable_RP_only));
|
|
|
1014
|
|
|
1015
|
|
|
1016 ###########################################################################
|
|
|
1017 ##### Printing output
|
|
|
1018
|
|
|
1019 #########################################
|
|
|
1020 # Get DNA info
|
|
|
1021 #########################################
|
|
|
1022 #print "PLUS_READS=$plus_Reads[0],$plus_Reads[1]\nMATE=$mate_chr,$mate_start,$INPUT_FASTA\n";
|
|
|
1023 $REF1_base = getSeq($plus_Reads[0], $plus_Reads[1], $INPUT_FASTA);
|
|
|
1024
|
|
|
1025 ### this is alt ref. for softclip its $plus_Reads[3]
|
|
|
1026 $REF2_base = getSeq($mate_chr, $mate_start, $INPUT_FASTA);
|
|
|
1027
|
|
|
1028 #########################################
|
|
|
1029 # print in VCF format
|
|
|
1030 #########################################
|
|
|
1031
|
|
|
1032 #### abs value to account for left and right reads.
|
|
|
1033 $isize = abs($plus_Reads[1]-$mate_start);
|
|
|
1034
|
|
|
1035 my $event_type=$type;
|
|
|
1036 $event_type=~ s/,|[0-9]//g;
|
|
|
1037 $INFO_1=join(";", "$sv_type", "EVENT=$event_type","END=$mate_start", "ISIZE=$isize","MATEID=$BND2_name");
|
|
|
1038 $INFO_2=join(";", "$sv_type", "EVENT=$event_type","END=$plus_Reads[1]", "ISIZE=$isize","MATEID=$BND1_name");
|
|
|
1039
|
|
|
1040 #### remove any white spaces.
|
|
|
1041 #### ask: did you mean to remove space from ends? eg. trim()
|
|
|
1042 $INFO_1=~s/\s//g;
|
|
|
1043 $INFO_2=~s/\s//g;
|
|
|
1044
|
|
|
1045 $FORMAT=$summary;
|
|
|
1046 $FORMAT=~ s/,|[0-9]//g;
|
|
|
1047 $FORMAT .= ":lSC:nSC:uRP:distl_levD";
|
|
|
1048 if($NO_MATE_SC){$INFO_2 .= ":NO_MATE_SC"}
|
|
|
1049 my $SAMPLE="0/1:";
|
|
|
1050 $SAMPLE .=$summary;
|
|
|
1051 # if($NO_MATE_SC){$SAMPLE.= ":$NO_MATE_SC"}
|
|
|
1052
|
|
|
1053 $SAMPLE=~s/[A-Z|,|_]//g;
|
|
|
1054 my $MATE_SAMPLE=$SAMPLE;
|
|
|
1055 $SAMPLE .= ":$lSC:$nSC:$num_unmapped_pairs:$distl_levD";
|
|
|
1056 $MATE_SAMPLE .=":NA:NA:NA:NA";
|
|
|
1057 $SAMPLE=~s/::/:/g;
|
|
|
1058 $MATE_SAMPLE=~s/::/:/g;
|
|
|
1059 $MATE_SAMPLE=~s/NA/0/g;
|
|
|
1060 $SAMPLE=~s/NA/0/g;
|
|
|
1061
|
|
|
1062 if($type !~ /INV/){
|
|
|
1063 $ALT_1 = join("","]",$mate_chr,":",$mate_start,"]",$REF1_base);
|
|
|
1064 $ALT_2 = join("",$REF2_base,"[",$plus_Reads[0],":",$plus_Reads[1],"[");
|
|
|
1065 # 2 321682 bnd_V T ]13:123456]T 6 PASS SVTYPE=BND
|
|
|
1066 # 13 123456 bnd_U C C[2:321682[ 6 PASS SVTYPE=BND
|
|
|
1067 } else {
|
|
|
1068 $ALT_1 = join("", "]", $plus_Reads[0], ":", $plus_Reads[1], "]", $REF2_base);
|
|
|
1069 $ALT_2 = join("", $REF1_base, "[", $mate_chr, ":", $mate_start, "[");
|
|
|
1070 }
|
|
|
1071
|
|
|
1072 if(($mate_chr) && ($plus_Reads[0])){
|
|
|
1073 print OUT join ("\t", $plus_Reads[0], $plus_Reads[1], $BND1_name, $REF1_base, $ALT_1, $QUAL,"PASS", $INFO_1, $FORMAT,$SAMPLE,"\n");
|
|
|
1074 print OUT join ("\t", $mate_chr, $mate_start, $BND2_name, $REF2_base, $ALT_2, $QUAL, "PASS", $INFO_2, $FORMAT,$MATE_SAMPLE,"\n");
|
|
|
1075 }
|
|
|
1076 }
|
|
|
1077
|
|
|
1078 ###############################################################################
|
|
|
1079 ###############################################################################
|
|
|
1080 sub get_counts_n_info {
|
|
|
1081 my ($event, $side, $mapQ, $file, $dist, $target, $upL, $lwL) = @_;
|
|
|
1082
|
|
|
1083 my $mate_info = "";
|
|
|
1084 my $cmd = "";
|
|
|
1085
|
|
|
1086 if ($event =~ /^CTX$/i) {
|
|
|
1087 #print "CTX side $side\n";
|
|
|
1088 if ($side =~ /right/i) {
|
|
|
1089 $cmd = qq{samtools view $new_blacklist -q $mapQ -f 32 -F 1536 $file $target};
|
|
|
1090 $cmd .= qq{ | perl -ane 'if(\$F[6] ne "="){\$end=\$F[7]+1; print join ("\\t",\$F[6],\$F[7],\$end,"\\n")}'};
|
|
|
1091 $cmd .= qq{ | sortBed | mergeBed -d $dist -n | sort -k4nr | head -1};
|
|
|
1092 #if($verbose){print "$cmd\n"}
|
|
|
1093 $mate_info=`$cmd`;
|
|
|
1094 } else {
|
|
|
1095 $cmd = qq{ samtools view $new_blacklist -q $mapQ -f 16 -F 1536 $file $target};
|
|
|
1096 $cmd .= qq{ | perl -ane 'if(\$F[6] ne "="){\$end=\$F[7]+1; print join ("\\t",\$F[6],\$F[7],\$end,"\\n")}'};
|
|
|
1097 $cmd .= qq{ | sortBed | mergeBed -d $dist -n | sort -k4nr | head -1};
|
|
|
1098 #if($verbose){print "$cmd\n"}
|
|
|
1099 $mate_info=`$cmd`;
|
|
|
1100 }
|
|
|
1101 } elsif ($event =~ /^DEL$/i) {
|
|
|
1102 #print "DEL side $side\n";
|
|
|
1103 if ($side =~ /right/i) {
|
|
|
1104 $cmd = qq{samtools view $new_blacklist -q $mapQ -f 32 -F 1552 $file $target};
|
|
|
1105 $cmd .= qq{ | awk '{OFS="\\t"}{if((\$7 ~ /=/)&&(\$9>$upL)){end=\$8+1;print \$3,\$8,end}}'};
|
|
|
1106 $cmd .= qq{ | sortBed | mergeBed -d $dist -n | sort -k4nr | head -1};
|
|
|
1107 #if($verbose){print "$cmd\n"}
|
|
|
1108 $mate_info=`$cmd`;
|
|
|
1109 } else {
|
|
|
1110 $cmd = qq{samtools view $new_blacklist -q $mapQ -F 1568 -f 16 $file $target};
|
|
|
1111 $cmd .= qq{ | awk '{OFS="\\t"} {if((\$7 ~ /=/)&&(\$9<-$upL)){end=\$8+1;print \$3,\$8,end}}'};
|
|
|
1112 $cmd .= qq{ | sortBed | mergeBed -d $dist_To_Soft -n | sort -k4nr | head -1};
|
|
|
1113 #if($verbose){print "$cmd\n"}
|
|
|
1114
|
|
|
1115 $mate_info=`$cmd`;
|
|
|
1116 }
|
|
|
1117 } elsif ($event =~ /^INS$/i) {
|
|
|
1118 #print "INS side $side\n";
|
|
|
1119 if ($side =~ /right/i) {
|
|
|
1120 $cmd = qq{samtools view $new_blacklist -q $mapQ -f 32 -F 1552 $file $target};
|
|
|
1121 $cmd .= qq{ | awk '{OFS="\\t"}{if((\$7 ~ /=/)&&(\$9<$lwL && \$9 > 0 )){end=\$8+1;print \$3,\$8,end}}'};
|
|
|
1122 $cmd .= qq{ | sortBed | mergeBed -d $dist -n | sort -k4nr | head -1};
|
|
|
1123 #if($verbose){print "$cmd\n"}
|
|
|
1124 $mate_info = `$cmd`;
|
|
|
1125 } else {
|
|
|
1126 $cmd = qq {samtools view $new_blacklist -q $mapQ -f 16 -F 1568 $file $target};
|
|
|
1127 $cmd .= qq{ | awk '{OFS="\\t"}{if((\$7 ~ /=/)&&(\$9>-$lwL && \$9 < 0 )){end=\$8+1;print \$3,\$8,end}}'};
|
|
|
1128 $cmd .= qq{ | sortBed | mergeBed -d $dist -n | sort -k4nr | head -1};
|
|
|
1129 #if($verbose){print "$cmd\n"}
|
|
|
1130 $mate_info = `$cmd`;
|
|
|
1131 }
|
|
|
1132 } elsif ($event =~ /^INV$/i) {
|
|
|
1133 #print "INV side $side\n";
|
|
|
1134 if ($side =~ /right/i) {
|
|
|
1135 $cmd = qq{samtools view $new_blacklist -q $mapQ -F 1596 $file $target};
|
|
|
1136 $cmd .= qq{ | awk '{OFS="\\t"}{if((\$7 ~ /=/)){end=\$8+1;print \$3,\$8,end}}'};
|
|
|
1137 $cmd .= qq{ | sortBed | mergeBed -d $dist_To_Soft -n | sort -k4nr | head -1};
|
|
|
1138 #if($verbose){print "$cmd\n"}
|
|
|
1139 $mate_info = `$cmd`;
|
|
|
1140 } else {
|
|
|
1141 $cmd = qq{samtools view $new_blacklist -q $mapQ -f 48 -F 1548 $file $target};
|
|
|
1142 $cmd .= qq{ | awk '{OFS="\\t"}{if((\$7 ~ /=/)){end=\$8+1;print \$3,\$8,end}}'};
|
|
|
1143 $cmd .= qq{ | sortBed | mergeBed -d $dist -n | sort -k4nr | head -1};
|
|
|
1144 #if($verbose){print "$cmd\n"}
|
|
|
1145 $mate_info = `$cmd`;
|
|
|
1146 }
|
|
|
1147 } elsif ($event =~ /^TDUP$/i) {
|
|
|
1148 #print "TDUP side $side\n";
|
|
|
1149 if ($side =~ /right/i) {
|
|
|
1150 $cmd = qq{samtools view $new_blacklist -q $mapQ -f 32 -F 1552 $file $target};
|
|
|
1151 # $cmd .= qq{ | awk '{OFS="\\t"}{if((\$7 ~ /=/)&&(\$9>$upL)){end=\$8+1;print \$3,\$8,end}}'};
|
|
|
1152 $cmd .= qq{ | awk '{OFS="\\t"}{if((\$7 ~ /=/)&&(\$4>\$8)&&(\$9<0)&& (\$9>$upL)){end=\$8+1;print \$3,\$8,end}}'};
|
|
|
1153 $cmd .= qq{ | sortBed | mergeBed -d $dist -n | sort -k4nr | head -1};
|
|
|
1154 #if($verbose){print "$cmd\n"}
|
|
|
1155 $mate_info = `$cmd`;
|
|
|
1156 } else {
|
|
|
1157 $cmd = qq{samtools view $new_blacklist -q $mapQ -f 16 -F 1568 $file $target};
|
|
|
1158 # $cmd .= qq{ | awk '{OFS="\\t"}{if((\$7 ~ /=/)&&(\$9<-$upL )){end=\$8+1;print \$3,\$8,end}}'};
|
|
|
1159 $cmd .= qq{ | awk '{OFS="\\t"}{if((\$7 ~ /=/)&&(\$4<\$8)&&(\$9>0)&&(\$9>$upL)){end=\$8+1;print \$3,\$8,end}}'};
|
|
|
1160 $cmd .= qq{ | sortBed | mergeBed -d $dist -n | sort -k4nr | head -1};
|
|
|
1161 #if($verbose){print "$cmd\n"}
|
|
|
1162 $mate_info = `$cmd`;
|
|
|
1163 }
|
|
|
1164 } elsif ($event =~ /^NOV_INS$/i) {
|
|
|
1165 #print "NOV_INS side $side\n";
|
|
|
1166 if ($side =~ /right/i) {
|
|
|
1167 $cmd = qq{samtools view $new_blacklist -q $mapQ -f 8 -F 1552 $file $target};
|
|
|
1168 $cmd .= qq{ | awk '{OFS="\\t"}{end=\$8+1;print \$3,\$8,end}'};
|
|
|
1169 $cmd .= qq{ | sortBed | mergeBed -d $dist -n | sort -k4nr | head -1};
|
|
|
1170 #if($verbose){print "$cmd\n"}
|
|
|
1171 $mate_info = `$cmd`;
|
|
|
1172 } else {
|
|
|
1173 $cmd = qq{samtools view $new_blacklist -q $mapQ -f 24 -F 1536 $file $target};
|
|
|
1174 $cmd .= qq{ | awk '{OFS="\\t"}{end=\$8+1;print \$3,\$8,end}'};
|
|
|
1175 $cmd .= qq{ | sortBed | mergeBed -d $dist -n | sort -k4nr | head -1};
|
|
|
1176 #if($verbose){print "$cmd\n"}
|
|
|
1177 $mate_info = `$cmd`;
|
|
|
1178 }
|
|
|
1179 }
|
|
|
1180
|
|
|
1181 $mate_info=~s/\n//g;
|
|
|
1182 my @tmp=split(/\t/, $mate_info);
|
|
|
1183
|
|
|
1184 my $counts = 0;
|
|
|
1185
|
|
|
1186 if (defined $tmp[3]) {
|
|
|
1187 $tmp[3] =~ s/\n//g;
|
|
|
1188
|
|
|
1189 $counts = $tmp[3] if (length($tmp[3]));
|
|
|
1190 }
|
|
|
1191 return ({count=>$counts, info=>$mate_info});
|
|
|
1192 }
|
