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1 #!/usr/bin/perl
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2 #Author Steven Hart, PhD
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3 #11-15-2012
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4 #Convert and filter BAM files into merged bed
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5 #Output should be
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6 #chrA StartA EndA chrB StartB EndB Gene_id #supportingReads StrandA StrandB
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7 #chr9 1000 5000 chr9 3000 3800 bedpe_example2 100 - +
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8
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9 use Cwd;
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10 use File::Basename;
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11 #Usage
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12 sub usage(){
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13 print "Usage: perl Bam2Pair.pl -b <BAM> -o <outfile>\n
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14 -isize [10000]\t\tThe insert size to be considered discordant\n
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15 -winsize [10000]\tThe distance between mate pairs to be considered the same\n
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16 -min [1]\t\tThe minimum number of reads required to support an SV event\n
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17 -prefix need a random prefix so files with the same name don't get created\n\n"
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18 ;
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19 }
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20 $bedtools=`which intersectBed`;
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21 $samtools=`which samtools`;
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22
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23 if(!defined($bedtools)){die "BEDtools must be installed\n";}
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24 if(!defined($samtools)){die "Samtools must be installed\n";}
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25 use Getopt::Long;
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26 #Declare variables
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27 GetOptions(
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28 'b=s' => \$BAM_FILE, #path to bam
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29 'out=s' => \$outfile, #path to output
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30 'java:s' => \$java ,
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31 'chrom:s' => \$chrom ,
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32 'isize=i' => \$isize,
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33 'winsize=i' => \$winsize,
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34 'prefix=s' => \$prefix,
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35 'min=i' => \$minSupport,
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36 'blacklist:s' => \$new_blacklist,
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37 'q=s' => \$qual,
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38 'v' => \$verbose
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39 );
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40 #if(defined($picard_path)){$picard_path=$picard_path} else {die "Must specify a path to PICARD so that files can be sorted and indexed properly\n"};
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41 if(!defined($BAM_FILE)){die "Must specify a BAM file!\n".usage();}
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42 if(!defined($outfile)){die "Must specify an out filename!\n".usage();}
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43 if(!defined($java)){$java=$java;}else{$java=`which java`}
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44 if(!defined($qual)){$qual=20}
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45 if($new_blacklist){$new_blacklist=" -L $new_blacklist"}
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46
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47
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48 $Filter_BAM=$BAM_FILE;
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49
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50 @bam=split("/",$Filter_BAM);
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51 $Filter_BAM=@bam[@bam-1];
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52 $Filter_BAM=~s/.bam/$prefix.bam/;
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53 $Filter_sam=$Filter_BAM;
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54 $Filter_sam=~s/.bam/.sam/;
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55
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56
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57
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58
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59 print "\nLooking for Discordant read pairs (and Unmated reads) without soft-clips\n";
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60
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61 #$command=join("","samtools view -h -q 20 -f 1 -F 1804 ",$BAM_FILE," ",$chrom," ",$new_blacklist," | awk -F\'\\t\' \'{if (\$9 > ", $isize, " || \$9 < -",$isize," || \$9 == 0 || \$1 ~ /^@/) print \$0}' > ",$Filter_sam);
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62
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63
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64 #Change command to allow reads where mate is unmapped & remove qual
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65 $command=join("","samtools view -h -f 1 -F 1800 -q $qual ",$BAM_FILE," ",$chrom," ",$new_blacklist," | awk -F\'\\t\' \'{if (\$9 > ", $isize, " || \$9 < -",$isize," || \$9 == 0 || \$1 ~ /^@/) print \$0}' > ",$Filter_sam);
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66
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67 print "$command\n" if ($verbose);
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68 system($command);
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69 $path = dirname(__FILE__);
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70 $Filter_cluster=$Filter_sam;
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71 $Filter_cluster=~s/.sam/.cluster/;
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72 $command=join("",$path,"/ReadCluster.pl -i=$Filter_sam -o=$Filter_cluster -m $minSupport");
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73 if($verbose){print "\n$command\n"};
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74
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75 system($command);
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76
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77 ##################################
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78 #Now there are 2 SAM files of filtered reads
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79 #.filter.cluster.inter.sam
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80 #.filter.cluster.intra.sam
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81 $result_pe=join("",$Filter_cluster,".out");
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82 $command=join("","cat ",$Filter_cluster,".int\*|perl -ane 'next if(\@F[0]=~/^\@/);if(\@F[6]!~/=/){print join(\"\\t\",\$F[11],\@F[2],\@F[3],\@F[6],\@F[7],\"\\n\")}else{print join(\"\\t\",\$F[11],\@F[2],\@F[3],\@F[2],\@F[7],\"\\n\")}' >",$result_pe);
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83 if($verbose){print $command."\n"};
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84 system($command);
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85 #my ($sample, $chrstart, $start, $chrend, $end)
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86 $command=join("","cat ",$result_pe," | ",$path,"/cluster.pair.pl ",$winsize," |awk '(\$6 >",$minSupport,")' >> ", $outfile);
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87 if($verbose){print $command."\n"};
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88 system($command);
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89 $filt1=join("",$Filter_cluster,".inter.sam");
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90 $filt2=join("",$Filter_cluster,".intra.sam");
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91
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92
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93 unlink($Filter_sam,$filt1,$filt2,$result_pe);
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94
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95 #########################################
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96 #Now determine if left or righ clipping surrogate
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97 print "\nBam2pair.pl Done\n";
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98
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