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1 #!/usr/bin/perl
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2 use strict;
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3
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4 ###Builds interpolated pileup from SAM file
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5 ##@description counts bases between paired ends and piles up single end reads.
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6 ##@output, uses a #header for the RNAME and then the number of reads per base
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7 ##@author sm8@sanger.ac.uk, Stephen B. Montgomery
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8
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9 ##@caveats
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10 ##Requires RNAME to have format as per example
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11 ## chromosome:NCBI36:18:1:76117153:1
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12 ## supercontig::NT_113883:1:137703:1
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13 ## clone::AC138827.3:1:149397:1
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14 ##Expects simple CIGAR characters, M, I and D
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15 ##Expects SAM file to be sorted.
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16 ##Expects 0x0010 to mark second read in PE file (as has been the observed case from MAQ output) (important for line 77)
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17
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18 ##Verify and read in SAM file
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19 my $sam_file = $ARGV[0];
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20 if(!defined($sam_file)) { die("No sam file defined on arg 1"); }
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21 unless(-f $sam_file) { die("Sam file does not exist: $sam_file"); }
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22 open(SAM, $sam_file) || die("Cannot open sam file");
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23
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24 ##Globals
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25 my $current_location = ""; ##Current RNAME being processed
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26 my $current_size = 0; ##Size of sequence region being processed
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27 my $current_position = 1; ##Current base being processed
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28 my $open = 0; ##Number of open reads (PE reads that have not been closed)
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29 my %close = (); ##Hash of closing positions, when the current_position gets to this position it subtracts the
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30 ##contained value from those open and deletes the indexed position from the hash
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31
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32 while (my $line = <SAM>) {
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33 my @tokens = split /\t/, $line;
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34
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35 if ($current_location ne $tokens[2]) { ##Start a new sequence region
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36 for (my $i = $current_position; $i <= $current_size; $i++) { ##Close the previous sequence region
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37 if (defined($close{$i})) {
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38 $open = $open - $close{$i};
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39 delete $close{$i};
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40 }
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41 print $open . "\n";
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42 }
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43 if ($current_location ne "") {
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44 print "\n";
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45 }
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46
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47 ##Initiate a new sequence region
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48 my @location_tokens = split /:/, $tokens[2];
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49 $current_position = 1;
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50 $current_location = $tokens[2];
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51 $current_size = $location_tokens[4];
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52 $open = 0;
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53 %close = ();
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54 print "#" . $tokens[2] . "\n";
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55
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56 ##Print pileup to just before the first read (will be 0)
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57 for (my $current_position = 1; $current_position < $tokens[3]; $current_position++) {
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58 print $open . "\n";
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59 }
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60 $current_position = $tokens[3];
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61
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62 } else { ##Sequence region already open
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63 if ($tokens[3] > $current_position) { ##If the new read's position is greater than the current position
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64 ##cycle through to catch up to the current position
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65 for (my $i = $current_position; $i < $tokens[3]; $i++) {
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66 if (defined($close{$i})) {
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67 $open = $open - $close{$i};
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68 delete $close{$i};
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69 }
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70 print $open . "\n";
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71 }
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72 $current_position = $tokens[3];
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73 }
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74 }
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75 $open++; ##Increment the number of open reads
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76
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77 if (($tokens[1] & 0x0080 || $tokens[1] & 0x0040) && $tokens[1] & 0x0010 && $tokens[1] & 0x0002) { ##if second read of mate pair, add close condition
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78 $open--;
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79 my $parsed_cig = &parseCigar($tokens[5]);
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80 my $seq_region_end = $tokens[3] + $parsed_cig->{'M'} + $parsed_cig->{'D'} - 1;
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81 if (!defined($close{$seq_region_end + 1})) { $close{$seq_region_end + 1} = 0; }
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82 $close{$seq_region_end + 1} = $close{$seq_region_end + 1} + 1;
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83 } elsif (!($tokens[1] & 0x0001) || !($tokens[1] & 0x0002)) { ##if unpaired, add close condition
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84 my $parsed_cig = &parseCigar($tokens[5]);
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85 my $seq_region_end = $tokens[3] + $parsed_cig->{'M'} + $parsed_cig->{'D'} - 1;
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86 if (!defined($close{$seq_region_end + 1})) { $close{$seq_region_end + 1} = 0; }
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87 $close{$seq_region_end + 1} = $close{$seq_region_end + 1} + 1;
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88 } else {
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89 #do nothing
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90 }
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91 }
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92 for (my $i = $current_position; $i <= $current_size; $i++) { ##Finish up the last sequence region
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93 if (defined($close{$i})) {
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94 $open = $open - $close{$i};
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95 delete $close{$i};
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96 }
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97 print $open . "\n";
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98 }
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99 print "\n";
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100 close(SAM);
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101 exit(0);
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102
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103 ##reads and tokenizes simple cigarline
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104 sub parseCigar() {
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105 my $cigar_line = shift;
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106 $cigar_line =~ s/([0-9]*[A-Z]{1})/$1\t/g;
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107 my @cigar_tokens = split /\t/, $cigar_line;
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108 my %parsed = ('M' => 0,
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109 'I' => 0,
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110 'D' => 0);
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111 my @events = ();
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112 for(my $i = 0; $i < scalar(@cigar_tokens); $i++) {
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113 if ($cigar_tokens[$i] =~ /([0-9]+)([A-Z]{1})/g) {
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114 if (!defined($parsed{$2})) { $parsed{$2} = 0; }
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115 my $nt = $2;
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116 if ($nt ne "M" && $nt ne "D" && $nt ne "I") { $nt = "M"; }
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117 $parsed{$nt} += $1;
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118 my %event_el = ("t" => $nt,
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119 "n" => $1);
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120 push @events, \%event_el;
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121 }
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122 }
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123 $parsed{'events'} = \@events;
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124 return \%parsed;
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125 }
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