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1 #! /usr/bin/perl -w
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2
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3 use strict;
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4 use POSIX;
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5
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6
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7 die "Usage: pileup_parser.pl <in_file> <ref_base_column> <read_bases_column> <base_quality_column> <coverage column> <qv cutoff> <coverage cutoff> <SNPs only?> <output bed?> <coord_column> <out_file> <total_diff> <print_qual_bases>\n" unless @ARGV == 13;
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8
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9 my $in_file = $ARGV[0];
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10 my $ref_base_column = $ARGV[1]-1; # 1 based
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11 my $read_bases_column = $ARGV[2]-1; # 1 based
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12 my $base_quality_column = $ARGV[3]-1; # 1 based
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13 my $cvrg_column = $ARGV[4]-1; # 1 based
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14 my $quality_cutoff = $ARGV[5]; # phred scale integer
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15 my $cvrg_cutoff = $ARGV[6]; # unsigned integer
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16 my $SNPs_only = $ARGV[7]; # set to "Yes" to print only positions with SNPs; set to "No" to pring everything
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17 my $bed = $ARGV[8]; #set to "Yes" to convert coordinates to bed format (0-based start, 1-based end); set to "No" to leave as is
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18 my $coord_column = $ARGV[9]-1; #1 based
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19 my $out_file = $ARGV[10];
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20 my $total_diff = $ARGV[11]; # set to "Yes" to print total number of deviant based
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21 my $print_qual_bases = $ARGV[12]; #set to "Yes" to print quality and read base columns
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22
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23 my $invalid_line_counter = 0;
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24 my $first_skipped_line = "";
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25 my %SNPs = ('A',0,'T',0,'C',0,'G',0);
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26 my $above_qv_bases = 0;
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27 my $SNPs_exist = 0;
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28 my $out_string = "";
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29 my $diff_count = 0;
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30
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31 open (IN, "<$in_file") or die "Cannot open $in_file $!\n";
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32 open (OUT, ">$out_file") or die "Cannot open $out_file $!\n";
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33
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34 while (<IN>) {
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35 chop;
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36 next if m/^\#/;
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37 my @fields = split /\t/;
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38 next if $fields[ $ref_base_column ] eq "*"; # skip indel lines
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39 my $read_bases = $fields[ $read_bases_column ];
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40 die "Coverage column" . ($cvrg_column+1) . " contains non-numeric values. Check your input parameters as well as format of input dataset." if ( not isdigit $fields[ $cvrg_column ] );
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41 next if $fields[ $cvrg_column ] < $cvrg_cutoff;
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42 my $base_quality = $fields[ $base_quality_column ];
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43 if ($read_bases =~ m/[\$\^\+-]/) {
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44 $read_bases =~ s/\^.//g; #removing the start of the read segement mark
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45 $read_bases =~ s/\$//g; #removing end of the read segment mark
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46 while ($read_bases =~ m/[\+-]{1}(\d+)/g) {
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47 my $indel_len = $1;
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48 $read_bases =~ s/[\+-]{1}$indel_len.{$indel_len}//; # remove indel info from read base field
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49 }
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50 }
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51 if ( length($read_bases) != length($base_quality) ) {
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52 $first_skipped_line = $. if $first_skipped_line eq "";
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53 ++$invalid_line_counter;
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54 next;
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55 }
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56 # after removing read block and indel data the length of read_base
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57 # field should identical to the length of base_quality field
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58
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59 my @bases = split //, $read_bases;
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60 my @qv = split //, $base_quality;
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61
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62 for my $base ( 0 .. @bases - 1 ) {
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63 if ( ord( $qv[ $base ] ) - 33 >= $quality_cutoff and $bases[ $base ] ne '*')
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64 {
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65 ++$above_qv_bases;
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66
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67 if ( $bases[ $base ] =~ m/[ATGC]/i )
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68 {
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69 $SNPs_exist = 1;
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70 $SNPs{ uc( $bases[ $base ] ) } += 1;
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71 $diff_count += 1;
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72 } elsif ( $bases[ $base ] =~ m/[\.,]/ ) {
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73 $SNPs{ uc( $fields[ $ref_base_column ] ) } += 1;
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74 }
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75 }
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76 }
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77
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78 if ($bed eq "Yes") {
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79 my $start = $fields[ $coord_column ] - 1;
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80 my $end = $fields[ $coord_column ];
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81 $fields[ $coord_column ] = "$start\t$end";
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82 }
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83
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84 if ($print_qual_bases ne "Yes") {
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85 $fields[ $base_quality_column ] = "";
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86 $fields[ $read_bases_column ] = "";
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87 }
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88
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89
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90 $out_string = join("\t", @fields); # \t$read_bases\t$base_quality";
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91 foreach my $SNP (sort keys %SNPs) {
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92 $out_string .= "\t$SNPs{$SNP}";
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93 }
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94
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95 if ($total_diff eq "Yes") {
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96 $out_string .= "\t$above_qv_bases\t$diff_count\n";
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97 } else {
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98 $out_string .= "\t$above_qv_bases\n";
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99 }
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100
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101 $out_string =~ s/\t+/\t/g;
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102
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103 if ( $SNPs_only eq "Yes" ) {
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104 print OUT $out_string if $SNPs_exist == 1;
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105 } else {
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106 print OUT $out_string;
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107 }
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108
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109
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110 %SNPs = ();
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111 %SNPs = ('A',0,'T',0,'C',0,'G',0);
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112 $above_qv_bases = 0;
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113 $SNPs_exist = 0;
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114 $diff_count = 0;
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115
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116
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117 }
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118
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119 print "Skipped $invalid_line_counter invalid line(s) beginning with line $first_skipped_line\n" if $invalid_line_counter > 0;
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120 close IN;
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121 close OUT;
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