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1 #!/usr/bin/perl -w
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2
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3 # Author: lh3
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4
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5 use strict;
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6 use warnings;
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7 use Getopt::Std;
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8
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9 my $version = '0.3.3';
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10 &usage if (@ARGV < 1);
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11
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12 my $command = shift(@ARGV);
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13 my %func = (showALEN=>\&showALEN, pileup2fq=>\&pileup2fq, varFilter=>\&varFilter, plp2vcf=>\&plp2vcf,
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14 unique=>\&unique, uniqcmp=>\&uniqcmp, sra2hdr=>\&sra2hdr, sam2fq=>\&sam2fq);
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15
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16 die("Unknown command \"$command\".\n") if (!defined($func{$command}));
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17 &{$func{$command}};
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18 exit(0);
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19
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20 #
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21 # showALEN
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22 #
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23
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24 sub showALEN {
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25 die(qq/Usage: samtools.pl showALEN <in.sam>\n/) if (@ARGV == 0 && -t STDIN);
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26 while (<>) {
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27 my @t = split;
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28 next if (/^\@/ || @t < 11);
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29 my $l = 0;
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30 $_ = $t[5];
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31 s/(\d+)[MI]/$l+=$1/eg;
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32 print join("\t", @t[0..5]), "\t$l\t", join("\t", @t[6..$#t]), "\n";
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33 }
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34 }
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35
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36 #
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37 # varFilter
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38 #
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39
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40 #
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41 # Filtration code:
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42 #
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43 # d low depth
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44 # D high depth
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45 # W too many SNPs in a window (SNP only)
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46 # G close to a high-quality indel (SNP only)
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47 # Q low RMS mapping quality (SNP only)
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48 # g close to another indel with higher quality (indel only)
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49 # s low SNP quality (SNP only)
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50 # i low indel quality (indel only)
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51
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52 sub varFilter {
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53 my %opts = (d=>3, D=>100, l=>30, Q=>25, q=>10, G=>25, s=>100, w=>10, W=>10, N=>2, p=>undef, S=>'', i=>'');
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54 getopts('pq:d:D:l:Q:w:W:N:G:S:i:', \%opts);
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55 die(qq/
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56 Usage: samtools.pl varFilter [options] <in.cns-pileup>
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57
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58 Options: -Q INT minimum RMS mapping quality for SNPs [$opts{Q}]
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59 -q INT minimum RMS mapping quality for gaps [$opts{q}]
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60 -d INT minimum read depth [$opts{d}]
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61 -D INT maximum read depth [$opts{D}]
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62 -S INT minimum SNP quality [$opts{S}]
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63 -i INT minimum indel quality [$opts{i}]
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64
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65 -G INT min indel score for nearby SNP filtering [$opts{G}]
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66 -w INT SNP within INT bp around a gap to be filtered [$opts{w}]
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67
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68 -W INT window size for filtering dense SNPs [$opts{W}]
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69 -N INT max number of SNPs in a window [$opts{N}]
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70
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71 -l INT window size for filtering adjacent gaps [$opts{l}]
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72
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73 -p print filtered variants
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74 \n/) if (@ARGV == 0 && -t STDIN);
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75
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76 # calculate the window size
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77 my ($ol, $ow, $oW) = ($opts{l}, $opts{w}, $opts{W});
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78 my $max_dist = $ol > $ow? $ol : $ow;
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79 $max_dist = $oW if ($max_dist < $oW);
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80 # the core loop
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81 my @staging; # (indel_filtering_score, flt_tag)
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82 while (<>) {
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83 my @t = split;
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84 next if (uc($t[2]) eq uc($t[3]) || $t[3] eq '*/*'); # skip non-var sites
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85 # clear the out-of-range elements
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86 while (@staging) {
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87 # Still on the same chromosome and the first element's window still affects this position?
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88 last if ($staging[0][3] eq $t[0] && $staging[0][4] + $staging[0][2] + $max_dist >= $t[1]);
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89 varFilter_aux(shift(@staging), $opts{p}); # calling a function is a bit slower, not much
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90 }
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91 my ($flt, $score) = (0, -1);
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92 # first a simple filter
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93 if ($t[7] < $opts{d}) {
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94 $flt = 2;
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95 } elsif ($t[7] > $opts{D}) {
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96 $flt = 3;
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97 }
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98 if ($t[2] eq '*') { # an indel
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99 if ($opts{i} && $opts{i}>$t[5]) { $flt = 8; }
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100 }
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101 elsif ($opts{S} && $opts{S}>$t[5]) { $flt = 7; } # SNP
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102
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103 # site dependent filters
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104 my $len=0;
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105 if ($flt == 0) {
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106 if ($t[2] eq '*') { # an indel
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107 # If deletion, remember the length of the deletion
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108 my ($a,$b) = split(m{/},$t[3]);
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109 my $alen = length($a) - 1;
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110 my $blen = length($b) - 1;
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111 if ( $alen>$blen )
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112 {
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113 if ( substr($a,0,1) eq '-' ) { $len=$alen; }
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114 }
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115 elsif ( substr($b,0,1) eq '-' ) { $len=$blen; }
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116
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117 $flt = 1 if ($t[6] < $opts{q});
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118 # filtering SNPs
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119 if ($t[5] >= $opts{G}) {
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120 for my $x (@staging) {
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121 # Is it a SNP and is it outside the SNP filter window?
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122 next if ($x->[0] >= 0 || $x->[4] + $x->[2] + $ow < $t[1]);
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123 $x->[1] = 5 if ($x->[1] == 0);
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124 }
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125 }
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126 # calculate the filtering score (different from indel quality)
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127 $score = $t[5];
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128 $score += $opts{s} * $t[10] if ($t[8] ne '*');
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129 $score += $opts{s} * $t[11] if ($t[9] ne '*');
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130 # check the staging list for indel filtering
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131 for my $x (@staging) {
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132 # Is it a SNP and is it outside the gap filter window
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133 next if ($x->[0] < 0 || $x->[4] + $x->[2] + $ol < $t[1]);
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134 if ($x->[0] < $score) {
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135 $x->[1] = 6;
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136 } else {
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137 $flt = 6; last;
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138 }
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139 }
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140 } else { # a SNP
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141 $flt = 1 if ($t[6] < $opts{Q});
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142 # check adjacent SNPs
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143 my $k = 1;
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144 for my $x (@staging) {
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145 ++$k if ($x->[0] < 0 && $x->[4] + $x->[2] + $oW >= $t[1] && ($x->[1] == 0 || $x->[1] == 4 || $x->[1] == 5));
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146 }
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147 # filtering is necessary
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148 if ($k > $opts{N}) {
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149 $flt = 4;
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150 for my $x (@staging) {
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151 $x->[1] = 4 if ($x->[0] < 0 && $x->[4] + $x->[2] + $oW >= $t[1] && $x->[1] == 0);
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152 }
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153 } else { # then check gap filter
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154 for my $x (@staging) {
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155 next if ($x->[0] < 0 || $x->[4] + $x->[2] + $ow < $t[1]);
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156 if ($x->[0] >= $opts{G}) {
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157 $flt = 5; last;
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158 }
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159 }
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160 }
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161 }
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162 }
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163 push(@staging, [$score, $flt, $len, @t]);
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164 }
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165 # output the last few elements in the staging list
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166 while (@staging) {
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167 varFilter_aux(shift @staging, $opts{p});
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168 }
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169 }
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170
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171 sub varFilter_aux {
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172 my ($first, $is_print) = @_;
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173 if ($first->[1] == 0) {
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174 print join("\t", @$first[3 .. @$first-1]), "\n";
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175 } elsif ($is_print) {
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176 print STDERR join("\t", substr("UQdDWGgsiX", $first->[1], 1), @$first[3 .. @$first-1]), "\n";
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177 }
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178 }
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179
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180 #
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181 # pileup2fq
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182 #
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183
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184 sub pileup2fq {
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185 my %opts = (d=>3, D=>255, Q=>25, G=>25, l=>10);
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186 getopts('d:D:Q:G:l:', \%opts);
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187 die(qq/
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188 Usage: samtools.pl pileup2fq [options] <in.cns-pileup>
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189
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190 Options: -d INT minimum depth [$opts{d}]
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191 -D INT maximum depth [$opts{D}]
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192 -Q INT min RMS mapQ [$opts{Q}]
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193 -G INT minimum indel score [$opts{G}]
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194 -l INT indel filter winsize [$opts{l}]\n
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195 /) if (@ARGV == 0 && -t STDIN);
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196
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197 my ($last_chr, $seq, $qual, @gaps, $last_pos);
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198 my $_Q = $opts{Q};
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199 my $_d = $opts{d};
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200 my $_D = $opts{D};
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201
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202 $last_chr = '';
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203 while (<>) {
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204 my @t = split;
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205 if ($last_chr ne $t[0]) {
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206 &p2q_post_process($last_chr, \$seq, \$qual, \@gaps, $opts{l}) if ($last_chr);
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207 $last_chr = $t[0];
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208 $last_pos = 0;
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209 $seq = ''; $qual = '';
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210 @gaps = ();
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211 }
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212 if ($t[1] - $last_pos != 1) {
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213 $seq .= 'n' x ($t[1] - $last_pos - 1);
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214 $qual .= '!' x ($t[1] - $last_pos - 1);
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215 }
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216 if ($t[2] eq '*') {
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217 push(@gaps, $t[1]) if ($t[5] >= $opts{G});
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218 } else {
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219 $seq .= ($t[6] >= $_Q && $t[7] >= $_d && $t[7] <= $_D)? uc($t[3]) : lc($t[3]);
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220 my $q = $t[4] + 33;
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221 $q = 126 if ($q > 126);
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222 $qual .= chr($q);
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223 }
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224 $last_pos = $t[1];
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225 }
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226 &p2q_post_process($last_chr, \$seq, \$qual, \@gaps, $opts{l});
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227 }
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228
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229 sub p2q_post_process {
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230 my ($chr, $seq, $qual, $gaps, $l) = @_;
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231 &p2q_filter_gaps($seq, $gaps, $l);
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232 print "\@$chr\n"; &p2q_print_str($seq);
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233 print "+\n"; &p2q_print_str($qual);
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234 }
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235
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236 sub p2q_filter_gaps {
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237 my ($seq, $gaps, $l) = @_;
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238 for my $g (@$gaps) {
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239 my $x = $g > $l? $g - $l : 0;
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240 substr($$seq, $x, $l + $l) = lc(substr($$seq, $x, $l + $l));
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241 }
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242 }
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243
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244 sub p2q_print_str {
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245 my ($s) = @_;
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246 my $l = length($$s);
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247 for (my $i = 0; $i < $l; $i += 60) {
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248 print substr($$s, $i, 60), "\n";
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249 }
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250 }
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251
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252 #
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253 # sam2fq
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254 #
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255
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256 sub sam2fq {
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257 my %opts = (n=>20, p=>'');
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258 getopts('n:p:', \%opts);
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259 die("Usage: samtools.pl sam2fq [-n 20] [-p <prefix>] <inp.sam>\n") if (@ARGV == 0 && -t STDIN);
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260 if ($opts{p} && $opts{n} > 1) {
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261 my $pre = $opts{p};
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262 my @fh;
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263 for (0 .. $opts{n}-1) {
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264 open($fh[$_], sprintf("| gzip > $pre.%.3d.fq.gz", $_)) || die;
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265 }
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266 my $i = 0;
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267 while (<>) {
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268 next if (/^@/);
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269 chomp;
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270 my @t = split("\t");
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271 next if ($t[9] eq '*');
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272 my ($name, $seq, $qual);
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273 if ($t[1] & 16) { # reverse strand
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274 $seq = reverse($t[9]);
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275 $qual = reverse($t[10]);
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276 $seq =~ tr/ACGTacgt/TGCAtgca/;
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277 } else {
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278 ($seq, $qual) = @t[9,10];
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279 }
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280 $name = $t[0];
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281 $name .= "/1" if ($t[1] & 0x40);
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282 $name .= "/2" if ($t[1] & 0x80);
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283 print {$fh[$i]} "\@$name\n$seq\n";
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284 if ($qual ne '*') {
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285 print {$fh[$i]} "+\n$qual\n";
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286 }
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287 $i = 0 if (++$i == $opts{n});
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288 }
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289 close($fh[$_]) for (0 .. $opts{n}-1);
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290 } else {
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291 die("To be implemented.\n");
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292 }
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293 }
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294
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295 #
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296 # sra2hdr
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297 #
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298
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299 # This subroutine does not use an XML parser. It requires that the SRA
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300 # XML files are properly formated.
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301 sub sra2hdr {
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302 my %opts = ();
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303 getopts('', \%opts);
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304 die("Usage: samtools.pl sra2hdr <SRA.prefix>\n") if (@ARGV == 0);
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305 my $pre = $ARGV[0];
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306 my $fh;
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307 # read sample
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308 my $sample = 'UNKNOWN';
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309 open($fh, "$pre.sample.xml") || die;
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310 while (<$fh>) {
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311 $sample = $1 if (/<SAMPLE.*alias="([^"]+)"/i);
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312 }
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313 close($fh);
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314 # read experiment
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315 my (%exp2lib, $exp);
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316 open($fh, "$pre.experiment.xml") || die;
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317 while (<$fh>) {
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318 if (/<EXPERIMENT.*accession="([^\s"]+)"/i) {
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319 $exp = $1;
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320 } elsif (/<LIBRARY_NAME>\s*(\S+)\s*<\/LIBRARY_NAME>/i) {
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321 $exp2lib{$exp} = $1;
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322 }
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323 }
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324 close($fh);
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325 # read run
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326 my ($run, @fn);
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327 open($fh, "$pre.run.xml") || die;
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328 while (<$fh>) {
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329 if (/<RUN.*accession="([^\s"]+)"/i) {
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330 $run = $1; @fn = ();
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331 } elsif (/<EXPERIMENT_REF.*accession="([^\s"]+)"/i) {
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332 print "\@RG\tID:$run\tSM:$sample\tLB:$exp2lib{$1}\n";
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333 } elsif (/<FILE.*filename="([^\s"]+)"/i) {
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334 push(@fn, $1);
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335 } elsif (/<\/RUN>/i) {
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336 if (@fn == 1) {
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337 print STDERR "$fn[0]\t$run\n";
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338 } else {
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339 for (0 .. $#fn) {
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340 print STDERR "$fn[$_]\t$run", "_", $_+1, "\n";
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341 }
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342 }
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343 }
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344 }
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345 close($fh);
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346 }
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347
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348 #
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349 # unique
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350 #
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351
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352 sub unique {
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353 my %opts = (f=>250.0, q=>5, r=>2, a=>1, b=>3);
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354 getopts('Qf:q:r:a:b:m', \%opts);
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355 die("Usage: samtools.pl unique [-f $opts{f}] <in.sam>\n") if (@ARGV == 0 && -t STDIN);
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356 my $last = '';
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357 my $recal_Q = !defined($opts{Q});
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358 my $multi_only = defined($opts{m});
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359 my @a;
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360 while (<>) {
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361 my $score = -1;
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362 print $_ if (/^\@/);
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363 $score = $1 if (/AS:i:(\d+)/);
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364 my @t = split("\t");
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365 next if (@t < 11);
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366 if ($score < 0) { # AS tag is unavailable
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367 my $cigar = $t[5];
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368 my ($mm, $go, $ge) = (0, 0, 0);
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369 $cigar =~ s/(\d+)[ID]/++$go,$ge+=$1/eg;
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370 $cigar = $t[5];
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371 $cigar =~ s/(\d+)M/$mm+=$1/eg;
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372 $score = $mm * $opts{a} - $go * $opts{q} - $ge * $opts{r}; # no mismatches...
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373 }
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374 $score = 1 if ($score < 1);
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375 if ($t[0] ne $last) {
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376 &unique_aux(\@a, $opts{f}, $recal_Q, $multi_only) if (@a);
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377 $last = $t[0];
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378 }
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379 push(@a, [$score, \@t]);
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380 }
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381 &unique_aux(\@a, $opts{f}, $recal_Q, $multi_only) if (@a);
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382 }
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383
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384 sub unique_aux {
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385 my ($a, $fac, $is_recal, $multi_only) = @_;
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386 my ($max, $max2, $max_i) = (0, 0, -1);
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387 for (my $i = 0; $i < @$a; ++$i) {
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388 if ($a->[$i][0] > $max) {
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389 $max2 = $max; $max = $a->[$i][0]; $max_i = $i;
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390 } elsif ($a->[$i][0] > $max2) {
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391 $max2 = $a->[$i][0];
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392 }
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393 }
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394 if ($is_recal) {
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395 if (!$multi_only || @$a > 1) {
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396 my $q = int($fac * ($max - $max2) / $max + .499);
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397 $q = 250 if ($q > 250);
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398 $a->[$max_i][1][4] = $q < 250? $q : 250;
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399 }
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400 }
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401 print join("\t", @{$a->[$max_i][1]});
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402 @$a = ();
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403 }
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404
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405 #
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406 # uniqcmp: compare two SAM files
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407 #
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408
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409 sub uniqcmp {
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410 my %opts = (q=>10, s=>100);
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411 getopts('pq:s:', \%opts);
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412 die("Usage: samtools.pl uniqcmp <in1.sam> <in2.sam>\n") if (@ARGV < 2);
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413 my ($fh, %a);
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414 warn("[uniqcmp] read the first file...\n");
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415 &uniqcmp_aux($ARGV[0], \%a, 0);
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416 warn("[uniqcmp] read the second file...\n");
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417 &uniqcmp_aux($ARGV[1], \%a, 1);
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418 warn("[uniqcmp] stats...\n");
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419 my @cnt;
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420 $cnt[$_] = 0 for (0..9);
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421 for my $x (keys %a) {
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422 my $p = $a{$x};
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423 my $z;
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424 if (defined($p->[0]) && defined($p->[1])) {
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425 $z = ($p->[0][0] == $p->[1][0] && $p->[0][1] eq $p->[1][1] && abs($p->[0][2] - $p->[1][2]) < $opts{s})? 0 : 1;
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426 if ($p->[0][3] >= $opts{q} && $p->[1][3] >= $opts{q}) {
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427 ++$cnt[$z*3+0];
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428 } elsif ($p->[0][3] >= $opts{q}) {
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429 ++$cnt[$z*3+1];
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430 } elsif ($p->[1][3] >= $opts{q}) {
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|
431 ++$cnt[$z*3+2];
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|
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432 }
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433 print STDERR "$x\t$p->[0][1]:$p->[0][2]\t$p->[0][3]\t$p->[0][4]\t$p->[1][1]:$p->[1][2]\t$p->[1][3]\t$p->[1][4]\t",
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434 $p->[0][5]-$p->[1][5], "\n" if ($z && defined($opts{p}) && ($p->[0][3] >= $opts{q} || $p->[1][3] >= $opts{q}));
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|
|
435 } elsif (defined($p->[0])) {
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|
|
436 ++$cnt[$p->[0][3]>=$opts{q}? 6 : 7];
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|
|
437 print STDERR "$x\t$p->[0][1]:$p->[0][2]\t$p->[0][3]\t$p->[0][4]\t*\t0\t*\t",
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|
|
438 $p->[0][5], "\n" if (defined($opts{p}) && $p->[0][3] >= $opts{q});
|
|
|
439 } else {
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|
|
440 print STDERR "$x\t*\t0\t*\t$p->[1][1]:$p->[1][2]\t$p->[1][3]\t$p->[1][4]\t",
|
|
|
441 -$p->[1][5], "\n" if (defined($opts{p}) && $p->[1][3] >= $opts{q});
|
|
|
442 ++$cnt[$p->[1][3]>=$opts{q}? 8 : 9];
|
|
|
443 }
|
|
|
444 }
|
|
|
445 print "Consistent (high, high): $cnt[0]\n";
|
|
|
446 print "Consistent (high, low ): $cnt[1]\n";
|
|
|
447 print "Consistent (low , high): $cnt[2]\n";
|
|
|
448 print "Inconsistent (high, high): $cnt[3]\n";
|
|
|
449 print "Inconsistent (high, low ): $cnt[4]\n";
|
|
|
450 print "Inconsistent (low , high): $cnt[5]\n";
|
|
|
451 print "Second missing (high): $cnt[6]\n";
|
|
|
452 print "Second missing (low ): $cnt[7]\n";
|
|
|
453 print "First missing (high): $cnt[8]\n";
|
|
|
454 print "First missing (low ): $cnt[9]\n";
|
|
|
455 }
|
|
|
456
|
|
|
457 sub uniqcmp_aux {
|
|
|
458 my ($fn, $a, $which) = @_;
|
|
|
459 my $fh;
|
|
|
460 $fn = "samtools view $fn |" if ($fn =~ /\.bam/);
|
|
|
461 open($fh, $fn) || die;
|
|
|
462 while (<$fh>) {
|
|
|
463 my @t = split;
|
|
|
464 next if (@t < 11);
|
|
|
465 # my $l = ($t[5] =~ /^(\d+)S/)? $1 : 0;
|
|
|
466 my $l = 0;
|
|
|
467 my ($x, $nm) = (0, 0);
|
|
|
468 $nm = $1 if (/NM:i:(\d+)/);
|
|
|
469 $_ = $t[5];
|
|
|
470 s/(\d+)[MI]/$x+=$1/eg;
|
|
|
471 @{$a->{$t[0]}[$which]} = (($t[1]&0x10)? 1 : 0, $t[2], $t[3]-$l, $t[4], "$x:$nm", $x - 4 * $nm);
|
|
|
472 }
|
|
|
473 close($fh);
|
|
|
474 }
|
|
|
475
|
|
|
476 sub plp2vcf {
|
|
|
477 while (<>) {
|
|
|
478 my @t = split;
|
|
|
479 next if ($t[3] eq '*/*');
|
|
|
480 if ($t[2] eq '*') { # indel
|
|
|
481 my @s = split("/", $t[3]);
|
|
|
482 my (@a, @b);
|
|
|
483 my ($ref, $alt);
|
|
|
484 for (@s) {
|
|
|
485 next if ($_ eq '*');
|
|
|
486 if (/^-/) {
|
|
|
487 push(@a, 'N'.substr($_, 1));
|
|
|
488 push(@b, 'N');
|
|
|
489 } elsif (/^\+/) {
|
|
|
490 push(@a, 'N');
|
|
|
491 push(@b, 'N'.substr($_, 1));
|
|
|
492 }
|
|
|
493 }
|
|
|
494 if ($a[0] && $a[1]) {
|
|
|
495 if (length($a[0]) < length($a[1])) {
|
|
|
496 $ref = $a[1];
|
|
|
497 $alt = ($b[0] . ('N' x (length($a[1]) - length($a[0])))) . ",$b[1]";
|
|
|
498 } elsif (length($a[0]) > length($a[1])) {
|
|
|
499 $ref = $a[0];
|
|
|
500 $alt = ($b[1] . ('N' x (length($a[0]) - length($a[1])))) . ",$b[0]";
|
|
|
501 } else {
|
|
|
502 $ref = $a[0];
|
|
|
503 $alt = ($b[0] eq $b[1])? $b[0] : "$b[0],$b[1]";
|
|
|
504 }
|
|
|
505 } else {
|
|
|
506 $ref = $a[0]; $alt = $b[0];
|
|
|
507 }
|
|
|
508 print join("\t", @t[0,1], '.', $ref, $alt, $t[5], '.', '.'), "\n";
|
|
|
509 } else { # SNP
|
|
|
510 }
|
|
|
511 }
|
|
|
512 }
|
|
|
513
|
|
|
514 #
|
|
|
515 # Usage
|
|
|
516 #
|
|
|
517
|
|
|
518 sub usage {
|
|
|
519 die(qq/
|
|
|
520 Program: samtools.pl (helper script for SAMtools)
|
|
|
521 Version: $version
|
|
|
522 Contact: Heng Li <lh3\@sanger.ac.uk>\n
|
|
|
523 Usage: samtools.pl <command> [<arguments>]\n
|
|
|
524 Command: varFilter filtering SNPs and short indels
|
|
|
525 pileup2fq generate fastq from `pileup -c'
|
|
|
526 showALEN print alignment length (ALEN) following CIGAR
|
|
|
527 \n/);
|
|
|
528 }
|
