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1 #!/usr/bin/env perl
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2
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3 =head1 LICENSE
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4
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5 Strelka Workflow Software
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6 Copyright (c) 2009-2013 Illumina, Inc.
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7
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8 This software is provided under the terms and conditions of the
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9 Illumina Open Source Software License 1.
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10
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11 You should have received a copy of the Illumina Open Source
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12 Software License 1 along with this program. If not, see
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13 <https://github.com/downloads/sequencing/licenses/>.
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14
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15 =head1 SYNOPSIS
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16
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17 filterSomaticVariants.pl [options] | --help
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18
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19 =head2 SUMMARY
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20
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21 Aggregate and filter the variant caller results by chromosome
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22
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23 =cut
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24
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25 use warnings FATAL => 'all';
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26 use strict;
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27
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28 use Carp;
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29 $SIG{__DIE__} = \&Carp::confess;
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30
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31 use File::Spec;
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32 use Getopt::Long;
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33 use Pod::Usage;
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34
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35 my $baseDir;
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36 my $libDir;
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37 #my $vcftDir;
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38 BEGIN {
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39 my $thisDir=(File::Spec->splitpath($0))[1];
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40 $baseDir=File::Spec->catdir($thisDir,File::Spec->updir());
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41 $libDir=File::Spec->catdir($baseDir,'lib');
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42 #my $optDir=File::Spec->catdir($baseDir,'opt');
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43 #$vcftDir=File::Spec->catdir($optDir,'vcftools','lib','perl5','site_perl');
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44 }
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45 use lib $libDir;
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46 use Utils;
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47 #use lib $vcftDir;
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48 #use Vcf;
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49
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50
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51 my $scriptName=(File::Spec->splitpath($0))[2];
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52 my $argCount=scalar(@ARGV);
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53 my $cmdline = join(' ',$0,@ARGV);
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54
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55
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56 my ($chrom, $configFile);
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57 my $help;
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58
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59 GetOptions( "chrom=s" => \$chrom,
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60 "config=s" => \$configFile,
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61 "help|h" => \$help) or pod2usage(2);
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62
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63 pod2usage(2) if($help);
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64 pod2usage(2) unless(defined($chrom));
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65 pod2usage(2) unless(defined($configFile));
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66
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67
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68
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69 #
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70 # read config and validate values
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71 #
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72 checkFile($configFile,"configuration ini");
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73 my $config = parseConfigIni($configFile);
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74
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75 my $chromSizeKey = "chrom_" . $chrom . "_size";
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76 my $chromKnownSizeKey = "chrom_" . $chrom . "_knownSize";
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77
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78 for (("outDir", $chromSizeKey, $chromKnownSizeKey)) {
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79 errorX("Undefined configuration option: '$_'") unless(defined($config->{derived}{$_}));
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80 }
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81 for (qw(binSize ssnvQuality_LowerBound sindelQuality_LowerBound
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82 isSkipDepthFilters depthFilterMultiple
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83 snvMaxFilteredBasecallFrac snvMaxSpanningDeletionFrac
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84 indelMaxRefRepeat indelMaxWindowFilteredBasecallFrac
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85 indelMaxIntHpolLength)) {
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86 errorX("Undefined configuration option: '$_'") unless(defined($config->{user}{$_}));
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87 }
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88
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89 my $outDir = $config->{derived}{outDir};
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90 my $chromDir = File::Spec->catdir($outDir,'chromosomes',$chrom);
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91 checkDir($outDir,"output");
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92 checkDir($chromDir,"output chromosome");
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93
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94 my $userconfig = $config->{user};
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95
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96 my $binList = getBinList($config->{derived}{$chromSizeKey},$userconfig->{binSize});
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97 for my $binId (@$binList) {
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98 my $dir = File::Spec->catdir($chromDir,'bins',$binId);
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99 checkDir($dir,"input bin");
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100 }
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101
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102 #
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103 # parameters from user config file:
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104 #
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105
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106 # minimum passed ssnv_nt Q-score:
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107 my $minQSSNT=$userconfig->{ssnvQuality_LowerBound};
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108 # minimum passed sindel_nt Q-score:
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109 my $minQSINT=$userconfig->{sindelQuality_LowerBound};
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110
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111 #
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112 # filtration parameters from user config file:
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113 #
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114
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115 # skip depth filters for targeted resequencing:
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116 my $isUseDepthFilter=(! $userconfig->{isSkipDepthFilters});
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117 # multiple of the normal mean coverage to filter snvs and indels
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118 my $depthFilterMultiple=$userconfig->{depthFilterMultiple};
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119 # max filtered basecall fraction for any sample
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120 my $snvMaxFilteredBasecallFrac=$userconfig->{snvMaxFilteredBasecallFrac};
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121 # max snv spanning-deletion fraction for any sample
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122 my $snvMaxSpanningDeletionFrac=$userconfig->{snvMaxSpanningDeletionFrac};
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123 # max indel reference repeat length
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124 my $indelMaxRefRepeat=$userconfig->{indelMaxRefRepeat};
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125 # max indel window filter fraction
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126 my $indelMaxWindowFilteredBasecallFrac=$userconfig->{indelMaxWindowFilteredBasecallFrac};
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127 # max indel interupted hompolymer length:
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128 my $indelMaxIntHpolLength=$userconfig->{indelMaxIntHpolLength};
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129
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130
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131 # first we want the normal sample mean chromosome depth:
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132 #
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133 my $filterCoverage;
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134 if($isUseDepthFilter) {
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135 my $totalCoverage = 0;
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136 for my $binId (@$binList) {
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137 my $sFile = File::Spec->catfile($chromDir,'bins',$binId,'strelka.stats');
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138 checkFile($sFile,"strelka bin stats");
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139 open(my $sFH, '<', $sFile)
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140 || errorX("Can't open file: '$sFile' $!");
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141
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142 my $is_found=0;
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143 while(<$sFH>) {
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144 next unless(/^NORMAL_NO_REF_N_COVERAGE\s/);
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145 my $is_bad = 0;
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146 if(not /sample_size:\s*(\d+)\s+/) { $is_bad=1; }
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147 my $ss=$1;
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148 # leave the regex for mean fairly loose to pick up scientific notation, etc..
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149 if(not /mean:\s*(\d[^\s]*|nan)\s+/) { $is_bad=1; }
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150 my $mean=$1;
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151 errorX("Unexpected format in file: '$sFile'") if($is_bad);
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152
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153 my $coverage = ( $ss==0 ? 0 : int($ss*$mean) );
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154
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155 $totalCoverage += $coverage;
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156 $is_found=1;
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157 last;
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158 }
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159 close($sFH);
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160 errorX("Unexpected format in file: '$sFile'") unless($is_found);
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161 }
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162
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163 my $chromKnownSize = $config->{derived}{$chromKnownSizeKey};
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164 my $normalMeanCoverage = ($totalCoverage/$chromKnownSize);
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165 $filterCoverage = $normalMeanCoverage*$depthFilterMultiple;
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166 }
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167
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168
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169 # add filter description to vcf header unless it already exists
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170 # return 1 if filter id already exists, client can decide if this is an error
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171 #
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172 sub add_vcf_filter($$$) {
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173 my ($vcf,$id,$desc) = @_;
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174 return 1 if(scalar(@{$vcf->get_header_line(key=>'FILTER', ID=>$id)}));
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175 $vcf->add_header_line({key=>'FILTER', ID=>$id,Description=>$desc});
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176 return 0;
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177 }
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178
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179
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180 sub check_vcf_for_sample($$$) {
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181 my ($vcf,$sample,$file) = @_;
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182 my $is_found=0;
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183 for ($vcf->get_samples()) {
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184 $is_found=1 if($_ eq $sample);
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185 }
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186 errorX("Failed to find sample '$sample' in vcf file '$file'") unless($is_found);
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187 }
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188
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189
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190
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191 my $depthFiltId="DP";
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192
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193
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194 # Runs all post-call vcf filters:
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195 #
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196 sub filterSnvFileList(\@$$$) {
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197 my ($ifiles,$depthFilterVal,$acceptFileName,$isUseDepthFilter) = @_;
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198
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199 my $baseFiltId="BCNoise";
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200 my $spanFiltId="SpanDel";
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201 my $qFiltId="QSS_ref";
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202
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203 open(my $aFH,'>',$acceptFileName)
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204 or errorX("Failed to open file: '$acceptFileName'");
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205
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206 my $is_first=1;
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207 for my $ifile (@$ifiles) {
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208 open(my $iFH,'<',"$ifile")
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209 or errorX("Failed to open file: '$ifile'");
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210 my $vcf = Vcf->new(fh=>$iFH);
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211
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212 # run some simple header validation on each vcf:
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213 $vcf->parse_header();
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214 check_vcf_for_sample($vcf,'NORMAL',$ifile);
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215 check_vcf_for_sample($vcf,'TUMOR',$ifile);
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216
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217 if($is_first) {
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218 # TODO: update vcf meta-data for chromosome-level filtered files
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219 #
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220 $vcf->remove_header_line(key=>"cmdline");
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221 $vcf->add_header_line({key=>"cmdline",value=>$cmdline});
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222 if($isUseDepthFilter) {
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223 $vcf->add_header_line({key=>"maxDepth_$chrom",value=>$depthFilterVal});
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224 add_vcf_filter($vcf,$depthFiltId,"Greater than ${depthFilterMultiple}x chromosomal mean depth in Normal sample");
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225 }
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226 add_vcf_filter($vcf,$baseFiltId,"Fraction of basecalls filtered at this site in either sample is at or above $snvMaxFilteredBasecallFrac");
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227 add_vcf_filter($vcf,$spanFiltId,"Fraction of reads crossing site with spanning deletions in either sample exceeeds $snvMaxSpanningDeletionFrac");
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228 add_vcf_filter($vcf,$qFiltId,"Normal sample is not homozygous ref or ssnv Q-score < $minQSSNT, ie calls with NT!=ref or QSS_NT < $minQSSNT");
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229 print $aFH $vcf->format_header();
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230 $is_first=0;
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231 }
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232
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233 while(my $x=$vcf->next_data_hash()) {
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234 my $norm=$x->{gtypes}->{NORMAL};
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235 my $tumr=$x->{gtypes}->{TUMOR};
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236
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237 my %filters;
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238
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239 # normal depth filter:
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240 my $normalDP=$norm->{DP};
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241 if($isUseDepthFilter) {
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242 $filters{$depthFiltId} = ($normalDP > $depthFilterVal);
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243 }
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244
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245 # filtered basecall fraction:
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246 my $normal_filt=($normalDP>0 ? $norm->{FDP}/$normalDP : 0);
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247
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248 my $tumorDP=$tumr->{DP};
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249 my $tumor_filt=($tumorDP>0 ? $tumr->{FDP}/$tumorDP : 0);
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250
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251 $filters{$baseFiltId}=(($normal_filt >= $snvMaxFilteredBasecallFrac) or
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252 ($tumor_filt >= $snvMaxFilteredBasecallFrac));
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253
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254 # spanning deletion fraction:
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255 my $normalSDP=$norm->{SDP};
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256 my $normalSpanTot=($normalDP+$normalSDP);
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257 my $normalSpanDelFrac=($normalSpanTot>0 ? ($normalSDP/$normalSpanTot) : 0);
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258
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259 my $tumorSDP=$tumr->{SDP};
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260 my $tumorSpanTot=($tumorDP+$tumorSDP);
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261 my $tumorSpanDelFrac=($tumorSpanTot>0 ? ($tumorSDP/$tumorSpanTot) : 0);
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262
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263 $filters{$spanFiltId}=(($normalSpanDelFrac > $snvMaxSpanningDeletionFrac) or
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264 ($tumorSpanDelFrac > $snvMaxSpanningDeletionFrac));
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265
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266 # Q-val filter:
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267 $filters{$qFiltId}=(($x->{INFO}->{NT} ne "ref") or
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268 ($x->{INFO}->{QSS_NT} < $minQSSNT));
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269
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270 $x->{FILTER} = $vcf->add_filter($x->{FILTER},%filters);
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271
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272 print $aFH $vcf->format_line($x);
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273 }
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274
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275 $vcf->close();
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276 close($iFH);
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277 }
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278
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279 close($aFH);
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280 }
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281
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282
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283
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284 sub updateA2(\@$) {
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285 my ($a2,$FH) = @_;
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286 my $line=<$FH>;
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287 unless(defined($line)) { errorX("Unexpected end of somatic indel window file"); }
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288 chomp $line;
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289 @$a2 = split("\t",$line);
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290 unless(scalar(@$a2)) { errorX("Unexpected format in somatic indel window file"); }
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291 }
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292
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293
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294
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295 sub filterIndelFileList(\@$$$) {
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296 my ($ifiles,$depthFilterVal,$acceptFileName,$isUseDepthFilter) = @_;
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297
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298 my $repeatFiltId="Repeat";
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299 my $iHpolFiltId="iHpol";
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300 my $baseFiltId="BCNoise";
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301 my $qFiltId="QSI_ref";
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302
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303 open(my $aFH,'>',$acceptFileName)
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304 or errorX("Failed to open file: '$acceptFileName'");
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305
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306 my $is_first=1;
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307 for my $ifile (@$ifiles) {
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308 open(my $iFH,'<',"$ifile")
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309 or errorX("Failed to open somatic indel file: '$ifile'");
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310
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311 my $iwfile = $ifile . ".window";
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312 open(my $iwFH,'<',"$iwfile")
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313 or errorX("Failed to open somatic indel window file: '$iwfile'");
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314
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315 my @a2; # hold window file data for one line in case we overstep...
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316
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317 my $vcf = Vcf->new(fh=>$iFH);
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318
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319 # run some simple header validation on each vcf:
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320 $vcf->parse_header();
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321 check_vcf_for_sample($vcf,'NORMAL',$ifile);
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322 check_vcf_for_sample($vcf,'TUMOR',$ifile);
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323
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324 if($is_first) {
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325 # TODO: update all vcf meta-data for chromosome-level filtered files
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326 #
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327 $vcf->remove_header_line(key=>"cmdline");
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328 $vcf->add_header_line({key=>"cmdline",value=>$cmdline});
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329 if($isUseDepthFilter) {
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330 $vcf->add_header_line({key=>"maxDepth_$chrom",value=>$depthFilterVal});
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331 }
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332 $vcf->add_header_line({key=>'FORMAT', ID=>'DP50',Number=>1,Type=>'Float',Description=>'Average tier1 read depth within 50 bases'});
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333 $vcf->add_header_line({key=>'FORMAT', ID=>'FDP50',Number=>1,Type=>'Float',Description=>'Average tier1 number of basecalls filtered from original read depth within 50 bases'});
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334 $vcf->add_header_line({key=>'FORMAT', ID=>'SUBDP50',Number=>1,Type=>'Float',Description=>'Average number of reads below tier1 mapping quality threshold aligned across sites within 50 bases'});
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335 if($isUseDepthFilter) {
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336 add_vcf_filter($vcf,$depthFiltId,"Greater than ${depthFilterMultiple}x chromosomal mean depth in Normal sample");
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337 }
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338 add_vcf_filter($vcf,$repeatFiltId,"Sequence repeat of more than ${indelMaxRefRepeat}x in the reference sequence");
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339 add_vcf_filter($vcf,$iHpolFiltId,"Indel overlaps an interupted homopolymer longer than ${indelMaxIntHpolLength}x in the reference sequence");
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340 add_vcf_filter($vcf,$baseFiltId,"Average fraction of filtered basecalls within 50 bases of the indel exceeds $indelMaxWindowFilteredBasecallFrac");
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341 add_vcf_filter($vcf,$qFiltId,"Normal sample is not homozygous ref or sindel Q-score < $minQSINT, ie calls with NT!=ref or QSI_NT < $minQSINT");
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342 print $aFH $vcf->format_header();
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343 $is_first=0;
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344 }
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345
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346 while(my $x=$vcf->next_data_hash()) {
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347 $vcf->add_format_field($x,'DP50');
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348 $vcf->add_format_field($x,'FDP50');
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349 $vcf->add_format_field($x,'SUBDP50');
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350
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351 my $norm=$x->{gtypes}->{NORMAL};
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352 my $tumr=$x->{gtypes}->{TUMOR};
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353
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354 my $chrom=$x->{CHROM};
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355 my $pos=int($x->{POS});
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356
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357 # get matching line from window file:
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358 while((scalar(@a2)<2) or
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359 (($a2[0] le $chrom) and (int($a2[1]) < $pos))) {
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360 updateA2(@a2,$iwFH);
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361 }
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362 unless(scalar(@a2) and ($a2[0] eq $chrom) and (int($a2[1]) == $pos))
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363 { errorX("Can't find somatic indel window position.\nIndel line: " . $vcf->format_line($x) ); }
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364
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365 # add window data to vcf record:
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366 #
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367 $norm->{DP50} = $a2[2]+$a2[3];
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368 $norm->{FDP50} = $a2[3];
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369 $norm->{SUBDP50} = $a2[4];
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370 $tumr->{DP50} = $a2[5]+$a2[6];
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371 $tumr->{FDP50} = $a2[6];
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372 $tumr->{SUBDP50} = $a2[7];
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373
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374 my %filters;
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375
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376 # normal depth filter:
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377 my $normalDP=$norm->{DP};
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378 if($isUseDepthFilter) {
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379 $filters{$depthFiltId}=($normalDP > $depthFilterVal);
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380 }
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381
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382 # ref repeat
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383 my $refRep=$x->{INFO}->{RC};
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384 $filters{$repeatFiltId}=(defined($refRep) and
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385 ($refRep > $indelMaxRefRepeat));
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386
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387 # ihpol
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388 my $iHpol=$x->{INFO}->{IHP};
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389 $filters{$iHpolFiltId}=(defined($iHpol) and
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390 ($iHpol > $indelMaxIntHpolLength));
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391
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392 # base filt:
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393 my $normWinFrac=( $norm->{DP50} ? $norm->{FDP50}/$norm->{DP50} : 0 );
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394 my $tumrWinFrac=( $tumr->{DP50} ? $tumr->{FDP50}/$tumr->{DP50} : 0 );
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395 $filters{$baseFiltId}=( ($normWinFrac >= $indelMaxWindowFilteredBasecallFrac) or
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396 ($tumrWinFrac >= $indelMaxWindowFilteredBasecallFrac) );
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397
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398 # Q-val filter:
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399 $filters{$qFiltId}=(($x->{INFO}->{NT} ne "ref") or
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400 ($x->{INFO}->{QSI_NT} < $minQSINT));
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401
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402 $x->{FILTER} = $vcf->add_filter($x->{FILTER},%filters);
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403
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404 print $aFH $vcf->format_line($x);
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405 }
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406
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407 $vcf->close();
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408 close($iFH);
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409 close($iwFH);
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410 }
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411
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412 close($aFH);
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413 }
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414
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415
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416
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417 my @ssnvFiles;
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418 my @sindelFiles;
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419 for my $binId (@$binList) {
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420 my $ssnvFile = File::Spec->catfile($chromDir,'bins',$binId,'somatic.snvs.unfiltered.vcf');
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421 my $sindelFile = File::Spec->catfile($chromDir,'bins',$binId,'somatic.indels.unfiltered.vcf');
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422 checkFile($ssnvFile,"bin snv file");
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423 checkFile($sindelFile,"bin indel file");
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424 push @ssnvFiles,$ssnvFile;
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425 push @sindelFiles,$sindelFile;
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426 }
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427
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428 my $ssnvOutFile = File::Spec->catfile($chromDir,"somatic.snvs.vcf");
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429 filterSnvFileList(@ssnvFiles,$filterCoverage,$ssnvOutFile,$isUseDepthFilter);
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430
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431 my $sindelOutFile = File::Spec->catfile($chromDir,"somatic.indels.vcf");
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432 filterIndelFileList(@sindelFiles,$filterCoverage,$sindelOutFile,$isUseDepthFilter);
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433
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434
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435 1;
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