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Chameleolyser.pl
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Chameleolyser.pl
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####################################################################################################################
####################################################################################################################
####################################################################################################################
####################################################################################################################
########## ##########
########## CHAMELEOLYSER ##########
########## ##########
####################################################################################################################
####################################################################################################################
####################################################################################################################
####################################################################################################################
##################################################################################################
##################################################################################################
##################################################################################################
####### Load Libraries #######
##################################################################################################
##################################################################################################
##################################################################################################
use Getopt::Long;
use IO::Zlib;
use strict;
use warnings;
use Data::Dumper;
use List::Util qw(min max);
##################################################################################################
##################################################################################################
##################################################################################################
####### Initialize & Declare #######
##################################################################################################
##################################################################################################
##################################################################################################
######################################################
### PROGRAM SETTINGS ###
######################################################
my $WORKING_DIR = "";
my $PREFIX = "";
my $OMIM = "";
my $SAMPLE_NAME = "";
my $ALIGNMENT_FP = "";
my $NR_OF_THREADS = 1;
######################################################
### PROGRAM OPTIONS ###
######################################################
my $PrepareBED = 0;
my $MaskReferenceGenome = 0;
my $GenerateMaskedAlignmentAndVcf = 0;
my $FilterRawVariants = 0;
##################################################################################################
##################################################################################################
##################################################################################################
####### Get Options #######
##################################################################################################
##################################################################################################
##################################################################################################
GetOptions ("WORKING_DIR=s" => \$WORKING_DIR,
"PREFIX=s" => \$PREFIX,
"OMIM=s" => \$OMIM,
"SAMPLE_NAME=s" => \$SAMPLE_NAME,
"ALIGNMENT_FP=s" => \$ALIGNMENT_FP,
"NR_OF_THREADS=i" => \$NR_OF_THREADS,
"PrepareBED" => \$PrepareBED,
"MaskReferenceGenome" => \$MaskReferenceGenome,
"GenerateMaskedAlignmentAndVcf" => \$GenerateMaskedAlignmentAndVcf,
"FilterRawVariants" => \$FilterRawVariants);
##################################################################################################
##################################################################################################
##################################################################################################
####### MAIN PROGRAM #######
##################################################################################################
##################################################################################################
##################################################################################################
PrepareBED ($WORKING_DIR,
$PREFIX,
$OMIM) if $PrepareBED;
MaskReferenceGenome ($WORKING_DIR,
$PREFIX,
$OMIM) if $MaskReferenceGenome;
GenerateMaskedAlignmentAndVcf ($WORKING_DIR,
$PREFIX,
$OMIM,
$SAMPLE_NAME,
$ALIGNMENT_FP,
$NR_OF_THREADS) if $GenerateMaskedAlignmentAndVcf;
FilterRawVariants ($WORKING_DIR,
$PREFIX,
$OMIM,
$SAMPLE_NAME) if $FilterRawVariants;
##################################################################################################
##################################################################################################
##################################################################################################
####### SUBROUTINES #######
##################################################################################################
##################################################################################################
##################################################################################################
sub PrepareBED {
(my $WORKING_DIR,
my $PREFIX,
my $OMIM)
= @_;
# test if working directory exists
if (! -d $WORKING_DIR){
print "$WORKING_DIR should already exist.\n";
print "Please enter an existing working directory.\n";
die("$!\n");
}
# download BEDs
unless (-d "$WORKING_DIR/BED/"){system("mkdir $WORKING_DIR/BED/");}
chdir ("$WORKING_DIR/BED/");
if ($PREFIX eq "chr"){
if ($OMIM eq "yes"){
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/All.formasking.omim.noalt.chr.bed");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/All.forextraction.omim.noalt.chr.bed");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/All.forvarcall.omim.noalt.chr.bed");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/All.homologousexons.omim.noalt.chr.bed");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/AllSDs.noalt.chr.bed");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/AllToMapOnToOther.chr.txt.gz");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/MappedSDs.chr.txt");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/PosToRegionID.chr.txt.gz");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/HPs.noalt.chr.bed.gz");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/SitesToExclude.noalt.chr.bed");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/CohortAlleleFreq.chr.txt");
system("gunzip HPs.noalt.chr.bed.gz");
}
else {
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/All.formasking.noalt.chr.bed");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/All.forextraction.noalt.chr.bed");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/All.forvarcall.noalt.chr.bed");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/All.homologousexons.noalt.chr.bed");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/AllSDs.noalt.chr.bed");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/AllToMapOnToOther.chr.txt.gz");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/MappedSDs.chr.txt");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/PosToRegionID.chr.txt.gz");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/HPs.noalt.chr.bed.gz");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/SitesToExclude.noalt.chr.bed");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/CohortAlleleFreq.chr.txt");
system("gunzip HPs.noalt.chr.bed.gz");
}
}
else {
if ($OMIM eq "yes"){
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/All.formasking.omim.noalt.bed");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/All.forextraction.omim.noalt.bed");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/All.forvarcall.omim.noalt.bed");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/All.homologousexons.omim.noalt.bed");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/AllSDs.noalt.bed");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/AllToMapOnToOther.txt.gz");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/MappedSDs.txt");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/PosToRegionID.txt.gz");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/HPs.noalt.bed.gz");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/SitesToExclude.noalt.bed");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/CohortAlleleFreq.txt");
system("gunzip HPs.noalt.bed.gz");
}
else {
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/All.formasking.noalt.bed");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/All.forextraction.noalt.bed");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/All.forvarcall.noalt.bed");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/All.homologousexons.noalt.bed");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/AllSDs.noalt.bed");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/AllToMapOnToOther.txt.gz");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/MappedSDs.txt");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/PosToRegionID.txt.gz");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/HPs.noalt.bed.gz");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/SitesToExclude.noalt.bed");
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/CohortAlleleFreq.txt");
system("gunzip HPs.noalt.bed.gz");
}
}
system ("wget https://raw.githubusercontent.com/Genome-Bioinformatics-RadboudUMC/ChameleolyserBEDs/main/RegionID_ToStrand.txt");
}
sub MaskReferenceGenome {
(my $WORKING_DIR,
my $PREFIX,
my $OMIM)
= @_;
# find Picard
my $PicardJarPath = `which picard`;
$PicardJarPath =~ s/\n//g;
$PicardJarPath =~ s/bin\/.*$/bin\//g;
$PicardJarPath .= "../share/picard-2.20.8-0/picard.jar";
# test if working directory exists
if (! -d $WORKING_DIR){
print "$WORKING_DIR should already exist.\n";
print "Please enter an existing working directory.\n";
die("$!\n");
}
# make new subdirs
unless(-d "$WORKING_DIR/REF/"){system("mkdir $WORKING_DIR/REF/");}
# download RefSeq #
chdir ("$WORKING_DIR/REF/");
if ($PREFIX eq "chr"){
if ($OMIM eq "yes"){
system ("wget https://ftp.ncbi.nlm.nih.gov/genomes/refseq/vertebrate_mammalian/Homo_sapiens/all_assembly_versions/GCF_000001405.25_GRCh37.p13/GRCh37_seqs_for_alignment_pipelines/GCA_000001405.14_GRCh37.p13_full_analysis_set.fna.gz");
system ("gunzip GCA_000001405.14_GRCh37.p13_full_analysis_set.fna.gz");
system ("mv GCA_000001405.14_GRCh37.p13_full_analysis_set.fna hg19.chr.fa");
system ("bwa index hg19.chr.fa");
system ("samtools faidx hg19.chr.fa");
system ("awk -v OFS='\t' {'print \$1,\$2'} hg19.chr.fa.fai > hg19.chr.txt");
system ("java -Xmx8G -jar $PicardJarPath CreateSequenceDictionary REFERENCE=hg19.chr.fa OUTPUT=hg19.chr.dict");
system ("bedtools maskfasta -fi hg19.chr.fa -bed ../BED/All.formasking.omim.noalt.chr.bed -fo hg19.masked.chr.fa");
system ("bwa index hg19.masked.chr.fa");
system ("samtools faidx hg19.masked.chr.fa");
system ("java -Xmx8G -jar $PicardJarPath CreateSequenceDictionary REFERENCE=hg19.masked.chr.fa OUTPUT=hg19.masked.chr.dict");
}
else {
system ("wget https://ftp.ncbi.nlm.nih.gov/genomes/refseq/vertebrate_mammalian/Homo_sapiens/all_assembly_versions/GCF_000001405.25_GRCh37.p13/GRCh37_seqs_for_alignment_pipelines/GCA_000001405.14_GRCh37.p13_full_analysis_set.fna.gz");
system ("gunzip GCA_000001405.14_GRCh37.p13_full_analysis_set.fna.gz");
system ("mv GCA_000001405.14_GRCh37.p13_full_analysis_set.fna hg19.chr.fa");
system ("bwa index hg19.chr.fa");
system ("samtools faidx hg19.chr.fa");
system ("awk -v OFS='\t' {'print \$1,\$2'} hg19.chr.fa.fai > hg19.chr.txt");
system ("java -Xmx8G -jar $PicardJarPath CreateSequenceDictionary REFERENCE=hg19.chr.fa OUTPUT=hg19.chr.dict");
system ("bedtools maskfasta -fi hg19.chr.fa -bed ../BED/All.formasking.noalt.chr.bed -fo hg19.masked.chr.fa");
system ("bwa index hg19.masked.chr.fa");
system ("samtools faidx hg19.masked.chr.fa");
system ("java -Xmx8G -jar $PicardJarPath CreateSequenceDictionary REFERENCE=hg19.masked.chr.fa OUTPUT=hg19.masked.chr.dict");
}
}
else {
if ($OMIM eq "yes"){
system ("wget http://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/human_g1k_v37.fasta.gz");
system ("gunzip human_g1k_v37.fasta.gz");
system ("mv human_g1k_v37.fasta hg19.fa");
system ("bwa index hg19.fa");
system ("samtools faidx hg19.fa");
system ("awk -v OFS='\t' {'print \$1,\$2'} hg19.fa.fai > hg19.txt");
system ("java -Xmx8G -jar $PicardJarPath CreateSequenceDictionary REFERENCE=hg19.fa OUTPUT=hg19.dict");
system ("bedtools maskfasta -fi hg19.fa -bed ../BED/All.formasking.omim.noalt.bed -fo hg19.masked.fa");
system ("bwa index hg19.masked.fa");
system ("samtools faidx hg19.masked.fa");
system ("java -Xmx8G -jar $PicardJarPath CreateSequenceDictionary REFERENCE=hg19.masked.fa OUTPUT=hg19.masked.dict");
}
else {
system ("wget http://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/human_g1k_v37.fasta.gz");
system ("gunzip human_g1k_v37.fasta.gz");
system ("mv human_g1k_v37.fasta hg19.fa");
system ("bwa index hg19.fa");
system ("samtools faidx hg19.fa");
system ("awk -v OFS='\t' {'print \$1,\$2'} hg19.fa.fai > hg19.txt");
system ("java -Xmx8G -jar $PicardJarPath CreateSequenceDictionary REFERENCE=hg19.fa OUTPUT=hg19.dict");
system ("bedtools maskfasta -fi hg19.fa -bed ../BED/All.formasking.noalt.bed -fo hg19.masked.fa");
system ("bwa index hg19.masked.fa");
system ("samtools faidx hg19.masked.fa");
system ("java -Xmx8G -jar $PicardJarPath CreateSequenceDictionary REFERENCE=hg19.masked.fa OUTPUT=hg19.masked.dict");
}
}
print "Masking Ready!\n";
}
sub GenerateMaskedAlignmentAndVcf {
(my $WORKING_DIR,
my $PREFIX,
my $OMIM,
my $SAMPLE_NAME,
my $ALIGNMENT_FP,
my $NR_OF_THREADS)
= @_;
# find Picard
my $PicardJarPath = `which picard`;
$PicardJarPath =~ s/\n//g;
$PicardJarPath =~ s/bin\/.*$/bin\//g;
$PicardJarPath .= "../share/picard-2.20.8-0/picard.jar";
my $ExtractionBedFP = "";
my $VarCallBedFP = "";
my $CovExonsFP = "";
my $RefGenome = "";
my $MaskedRefGenome = "";
my $GenomeFileFilePath = "";
if ($PREFIX eq "chr"){
if ($OMIM eq "yes"){
$ExtractionBedFP = "$WORKING_DIR/BED/All.forextraction.omim.noalt.chr.bed";
$VarCallBedFP = "$WORKING_DIR/BED/All.forvarcall.omim.noalt.chr.bed";
$CovExonsFP = "$WORKING_DIR/BED/All.homologousexons.omim.noalt.chr.bed";
$RefGenome = "$WORKING_DIR/REF/hg19.chr.fa";
$MaskedRefGenome = "$WORKING_DIR/REF/hg19.masked.chr.fa";
$GenomeFileFilePath = "$WORKING_DIR/REF/hg19.chr.txt";
}
else {
$ExtractionBedFP = "$WORKING_DIR/BED/All.forextraction.noalt.chr.bed";
$VarCallBedFP = "$WORKING_DIR/BED/All.forvarcall.noalt.chr.bed";
$CovExonsFP = "$WORKING_DIR/BED/All.homologousexons.noalt.chr.bed";
$RefGenome = "$WORKING_DIR/REF/hg19.chr.fa";
$MaskedRefGenome = "$WORKING_DIR/REF/hg19.masked.chr.fa";
$GenomeFileFilePath = "$WORKING_DIR/REF/hg19.chr.txt";
}
}
else {
if ($OMIM eq "yes"){
$ExtractionBedFP = "$WORKING_DIR/BED/All.forextraction.omim.noalt.bed";
$VarCallBedFP = "$WORKING_DIR/BED/All.forvarcall.omim.noalt.bed";
$CovExonsFP = "$WORKING_DIR/BED/All.homologousexons.omim.noalt.bed";
$RefGenome = "$WORKING_DIR/REF/hg19.fa";
$MaskedRefGenome = "$WORKING_DIR/REF/hg19.masked.fa";
$GenomeFileFilePath = "$WORKING_DIR/REF/hg19.txt";
}
else {
$ExtractionBedFP = "$WORKING_DIR/BED/All.forextraction.noalt.bed";
$VarCallBedFP = "$WORKING_DIR/BED/All.forvarcall.noalt.bed";
$CovExonsFP = "$WORKING_DIR/BED/All.homologousexons.noalt.bed";
$RefGenome = "$WORKING_DIR/REF/hg19.fa";
$MaskedRefGenome = "$WORKING_DIR/REF/hg19.masked.fa";
$GenomeFileFilePath = "$WORKING_DIR/REF/hg19.txt";
}
}
# test if working directory, refseq and BEDs exists
if (! -d $WORKING_DIR){
print "$WORKING_DIR should already exist.\n";
print "Please enter an existing working directory.\n";
die("$!\n");
}
if ( ! -f $ExtractionBedFP ||
! -f $VarCallBedFP ||
! -f $CovExonsFP ||
! -f $RefGenome ||
! -f $MaskedRefGenome ||
! -f $GenomeFileFilePath){
print "BEDs and RefSeqs should already be present\n";
print "Please first run PrepareBED and MaskReferenceGenome\n";
die("$!\n");
}
# Generate masked alignment
unless (-d "$WORKING_DIR/RAW/$SAMPLE_NAME/"){system("mkdir -p $WORKING_DIR/RAW/$SAMPLE_NAME/");};
chdir ("$WORKING_DIR/RAW/$SAMPLE_NAME/");
system ("samtools view -b -L $ExtractionBedFP -o $SAMPLE_NAME.ori.bam $ALIGNMENT_FP");
system ("java -Xmx8G -jar $PicardJarPath SortSam I=$SAMPLE_NAME.ori.bam O=$SAMPLE_NAME.ori.sorted.bam SO=coordinate");
system ("java -Xmx8G -jar $PicardJarPath MarkDuplicates I=$SAMPLE_NAME.ori.sorted.bam O=$SAMPLE_NAME.ori.sorted.remdup.bam M=$SAMPLE_NAME.ori.sorted.remdup.met REMOVE_DUPLICATES=true");
system ("rm -f $SAMPLE_NAME.ori.sorted.bam");
system ("samtools index $SAMPLE_NAME.ori.sorted.remdup.bam");
system ("samtools view -bh -q 1 $SAMPLE_NAME.ori.sorted.remdup.bam > $SAMPLE_NAME.ori.sorted.remdup.uniq.bam");
system ("bedtools coverage -a $CovExonsFP -b $SAMPLE_NAME.ori.sorted.remdup.bam -counts | bgzip -c > $SAMPLE_NAME.ori.sorted.remdup.cov.gz");
system ("bedtools coverage -a $CovExonsFP -b $SAMPLE_NAME.ori.sorted.remdup.uniq.bam -counts | bgzip -c > $SAMPLE_NAME.ori.sorted.remdup.uniq.cov.gz");
system ("lofreq call --call-indels --force-overwrite --no-default-filter --use-orphan -a 1 -b 20 -B -N -f $RefGenome -l $VarCallBedFP -o $SAMPLE_NAME.ori.sorted.remdup.lofreq.vcf $SAMPLE_NAME.ori.sorted.remdup.bam");
system ("bgzip -c $SAMPLE_NAME.ori.sorted.remdup.lofreq.vcf > $SAMPLE_NAME.ori.sorted.remdup.lofreq.vcf.gz");
system ("tabix -p vcf $SAMPLE_NAME.ori.sorted.remdup.lofreq.vcf.gz");
system ("rm -f $SAMPLE_NAME.ori.sorted.remdup.lofreq.vcf");
system ("gatk --java-options \-Xmx4g\ HaplotypeCaller -R $RefGenome -I $SAMPLE_NAME.ori.sorted.remdup.bam -O $SAMPLE_NAME.ori.sorted.remdup.gatk.vcf -L $VarCallBedFP --annotation MappingQualityRankSumTest --annotation MappingQualityZero --annotation QualByDepth --annotation ReadPosRankSumTest --annotation RMSMappingQuality --annotation BaseQualityRankSumTest --annotation FisherStrand --annotation MappingQuality --annotation DepthPerAlleleBySample");
system ("bgzip -c $SAMPLE_NAME\.ori.sorted.remdup.gatk.vcf > $SAMPLE_NAME.ori.sorted.remdup.gatk.vcf.gz");
system ("tabix -p vcf $SAMPLE_NAME\.ori.sorted.remdup.gatk.vcf.gz");
system ("rm -f $SAMPLE_NAME\.ori.sorted.remdup.gatk.vcf");
system ("rm -f $SAMPLE_NAME\.ori.sorted.remdup.gatk.vcf.idx");
system ("bedtools coverage -a $VarCallBedFP -b $SAMPLE_NAME.ori.sorted.remdup.bam -d -sorted -g $GenomeFileFilePath > $SAMPLE_NAME.ori.sorted.remdup.cov");
system ("bgzip -c $SAMPLE_NAME.ori.sorted.remdup.cov > $SAMPLE_NAME.ori.sorted.remdup.cov.gz");
system ("tabix -p bed $SAMPLE_NAME.ori.sorted.remdup.cov.gz");
system ("rm -f $SAMPLE_NAME.ori.sorted.remdup.cov");
system ("samtools collate -uO $SAMPLE_NAME.ori.sorted.remdup.bam $SAMPLE_NAME | samtools fastq -1 $SAMPLE_NAME.1.fastq -2 $SAMPLE_NAME.2.fastq -0 $SAMPLE_NAME.0.fastq -t -");
system ("gzip $SAMPLE_NAME.1.fastq");
system ("gzip $SAMPLE_NAME.2.fastq");
system ("repair.sh -in1=$SAMPLE_NAME.1.fastq.gz in2=$SAMPLE_NAME.2.fastq.gz out1=$SAMPLE_NAME.rep.1.fastq.gz out2=$SAMPLE_NAME.rep.2.fastq.gz outsingle=$SAMPLE_NAME.rep.0.fastq.gz usejni=t");
system ("rm -f $SAMPLE_NAME.1.fastq.gz");
system ("rm -f $SAMPLE_NAME.2.fastq.gz");
system ("rm -f $SAMPLE_NAME.0.fastq");
system ("bwa mem -t $NR_OF_THREADS -M $MaskedRefGenome $SAMPLE_NAME.rep.1.fastq.gz $SAMPLE_NAME.rep.2.fastq.gz > $SAMPLE_NAME.masked.sam");
system ("samtools view -Sb $SAMPLE_NAME.masked.sam > $SAMPLE_NAME.masked.bam");
system ("java -Xmx8G -jar $PicardJarPath SortSam I=$SAMPLE_NAME.masked.bam O=$SAMPLE_NAME.masked.sorted.bam SO=coordinate");
system ("rm -f $SAMPLE_NAME.rep.0.fastq.gz");
system ("rm -f $SAMPLE_NAME.rep.1.fastq.gz");
system ("rm -f $SAMPLE_NAME.rep.2.fastq.gz");
system ("rm -f $SAMPLE_NAME.masked.sam");
system ("rm -f $SAMPLE_NAME.masked.bam");
system ("java -Xmx8G -jar $PicardJarPath MarkDuplicates I=$SAMPLE_NAME.masked.sorted.bam O=$SAMPLE_NAME.masked.sorted.remdup.bam M=$SAMPLE_NAME.masked.sorted.remdup.met REMOVE_DUPLICATES=true");
system ("rm -f $SAMPLE_NAME.masked.sorted.bam");
system ("lofreq indelqual --dindel -f $MaskedRefGenome -o $SAMPLE_NAME.masked.sorted.remdup.bqsr.bam $SAMPLE_NAME.masked.sorted.remdup.bam");
system ("rm -f $SAMPLE_NAME.masked.sorted.remdup.bam");
system ("samtools index $SAMPLE_NAME.masked.sorted.remdup.bqsr.bam");
system ("java -Xmx8G -jar $PicardJarPath AddOrReplaceReadGroups I=$SAMPLE_NAME.masked.sorted.remdup.bqsr.bam O=$SAMPLE_NAME.masked.sorted.remdup.bqsr.rg.bam RGID=1 RGLB=lib1 RGPL=illumina RGPU=unit1 RGSM=$SAMPLE_NAME");
system ("rm -f $SAMPLE_NAME.masked.sorted.remdup.bqsr.bam");
system ("rm -f $SAMPLE_NAME.masked.sorted.remdup.bqsr.bam.bai");
system ("samtools index $SAMPLE_NAME.masked.sorted.remdup.bqsr.rg.bam");
system ("lofreq call --call-indels --force-overwrite --no-default-filter --use-orphan -a 1 -b 20 -B -N -f $MaskedRefGenome -l $VarCallBedFP -o $SAMPLE_NAME.masked.sorted.remdup.bqsr.rg.lofreq.vcf $SAMPLE_NAME.masked.sorted.remdup.bqsr.rg.bam");
system ("bgzip -c $SAMPLE_NAME.masked.sorted.remdup.bqsr.rg.lofreq.vcf > $SAMPLE_NAME.masked.sorted.remdup.bqsr.rg.lofreq.vcf.gz");
system ("tabix -p vcf $SAMPLE_NAME.masked.sorted.remdup.bqsr.rg.lofreq.vcf.gz");
system ("rm -f $SAMPLE_NAME.masked.sorted.remdup.bqsr.rg.lofreq.vcf");
system ("gatk --java-options \-Xmx4g\ HaplotypeCaller -R $MaskedRefGenome -I $SAMPLE_NAME.masked.sorted.remdup.bqsr.rg.bam -O $SAMPLE_NAME.masked.sorted.remdup.bqsr.rg.gatk.vcf -L $VarCallBedFP --annotation MappingQualityRankSumTest --annotation MappingQualityZero --annotation QualByDepth --annotation ReadPosRankSumTest --annotation RMSMappingQuality --annotation BaseQualityRankSumTest --annotation FisherStrand --annotation MappingQuality --annotation DepthPerAlleleBySample");
system ("bgzip -c $SAMPLE_NAME.masked.sorted.remdup.bqsr.rg.gatk.vcf > $SAMPLE_NAME.masked.sorted.remdup.bqsr.rg.gatk.vcf.gz");
system ("tabix -p vcf $SAMPLE_NAME.masked.sorted.remdup.bqsr.rg.gatk.vcf.gz");
system ("rm -f $SAMPLE_NAME.masked.sorted.remdup.bqsr.rg.gatk.vcf");
system ("rm -f $SAMPLE_NAME.masked.sorted.remdup.bqsr.rg.gatk.vcf.idx");
system ("bedtools coverage -a $VarCallBedFP -b $SAMPLE_NAME.masked.sorted.remdup.bqsr.rg.bam -d -sorted -g $GenomeFileFilePath > $SAMPLE_NAME.masked.sorted.remdup.bqsr.rg.cov");
system ("bgzip -c $SAMPLE_NAME.masked.sorted.remdup.bqsr.rg.cov > $SAMPLE_NAME.masked.sorted.remdup.bqsr.rg.cov.gz");
system ("tabix -p bed $SAMPLE_NAME.masked.sorted.remdup.bqsr.rg.cov.gz");
system ("rm -f $SAMPLE_NAME.masked.sorted.remdup.bqsr.rg.cov");
}
sub FilterRawVariants {
(my $WORKING_DIR,
my $PREFIX,
my $OMIM,
my $SAMPLE_NAME)
= @_;
my $SDsBedFP = "";
my $AllToMapOnToOtherFP = "";
my $MappedSDsFP = "";
my $PositionToRegionFP = "";
my $HPsBedFP = "";
my $TroubleSitesFP = "";
my $CohortAF_FP = "";
my $RegionToStrandsFP = "$WORKING_DIR\/BED/RegionID_ToStrand.txt";
my $VariantsOutFP = $WORKING_DIR . "RAW/$SAMPLE_NAME/$SAMPLE_NAME.RawSNVs.txt";
my $VariantsFilter1OutFP = $WORKING_DIR . "RAW/$SAMPLE_NAME/$SAMPLE_NAME.Filter1SNVs.txt";
my $VariantsFilter2OutFP = $WORKING_DIR . "RAW/$SAMPLE_NAME/$SAMPLE_NAME.Filter2SNVs.txt";
my $VariantsFilter1BedFP = $WORKING_DIR . "RAW/$SAMPLE_NAME/$SAMPLE_NAME.Filter1SNVs.bed";
my $SortedVariantsFilter1BedFP = $WORKING_DIR . "RAW/$SAMPLE_NAME/$SAMPLE_NAME.Filter1SNVs.sorted.bed";
my $VariantsFilter1HpAnnoBedFP = $WORKING_DIR . "RAW/$SAMPLE_NAME/$SAMPLE_NAME.Filter1SNVs.HpAnno.bed";
my $FileHandle = new IO::Zlib;
my %SDsBED = ();
my %SDsVAR = ();
my @VCFs = ( "$SAMPLE_NAME.masked.sorted.remdup.bqsr.rg.lofreq.vcf.gz",
"$SAMPLE_NAME.ori.sorted.remdup.gatk.vcf.gz");
my $CovFilePath = $WORKING_DIR . "RAW/$SAMPLE_NAME/$SAMPLE_NAME.masked.sorted.remdup.bqsr.rg.cov.gz";
my %LoFreq_Variants = ();
my %GATK_Variants = ();
my %PosOfInterest = ();
my %CoordinateMappings = ();
my %Coverage = ();
my %MappedSDs = ();
my %PosToRegionName = ();
my %Strands = ();
my %VarsToExclude = ();
my %F1Variants = ();
my %SDtroublePos = ();
my %PopFreq = ();
if ($PREFIX eq "chr"){
$SDsBedFP = "$WORKING_DIR/BED/AllSDs.noalt.chr.bed";
$AllToMapOnToOtherFP = "$WORKING_DIR\/BED/AllToMapOnToOther.chr.txt.gz";
$MappedSDsFP = "$WORKING_DIR\/BED/MappedSDs.chr.txt";
$PositionToRegionFP = "$WORKING_DIR\/BED/PosToRegionID.chr.txt.gz";
$HPsBedFP = "$WORKING_DIR\/BED/HPs.noalt.chr.bed";
$TroubleSitesFP = "$WORKING_DIR\/BED/SitesToExclude.noalt.chr.bed";
$CohortAF_FP = "$WORKING_DIR\/BED/CohortAlleleFreq.chr.txt";
}
else {
$SDsBedFP = "$WORKING_DIR/BED/AllSDs.noalt.bed";
$AllToMapOnToOtherFP = "$WORKING_DIR\/BED/AllToMapOnToOther.txt.gz";
$MappedSDsFP = "$WORKING_DIR\/BED/MappedSDs.txt";
$PositionToRegionFP = "$WORKING_DIR\/BED/PosToRegionID.txt.gz";
$HPsBedFP = "$WORKING_DIR\/BED/HPs.noalt.bed";
$TroubleSitesFP = "$WORKING_DIR\/BED/SitesToExclude.noalt.bed";
$CohortAF_FP = "$WORKING_DIR\/BED/CohortAlleleFreq.txt";
}
# Read in SDs #
print "Read in SDs\n";
open SD, "$SDsBedFP" or die ("Can't open $SDsBedFP\n");
while(<SD>){
$_ =~ s/\n//g;
$_ =~ s/\r//g;
(my $Chrom, undef, undef, my $SD) = split (m/\t/, $_);
(undef, my $VarStart, undef, undef) = split(m/~/, $SD);
$SDsBED{$Chrom}{$VarStart}{$SD} = undef;
$SDsVAR{$SD} = undef;
}
close SD;
# Read In coorindate mappings #
print "Read In coorindate mappings\n";
$FileHandle = new IO::Zlib;
if ($FileHandle->open($AllToMapOnToOtherFP, "rb")){
while (<$FileHandle>){
$_ =~ s/\n//g;
(my $ToMapOnCoord, my $OtherCoord) = split(m/\t/, $_);
$CoordinateMappings{$ToMapOnCoord}{$OtherCoord} = undef;
}
}
# Read in mapped SDs
print "Read in mapped SDs\n";
open M, "$MappedSDsFP" or die ("Can't open $MappedSDsFP\n");
while (<M>){
$_ =~ s/\n//g;
(my $ToMapOnSD, my $OtherSD) = split (m/\t/, $_);
$MappedSDs{$ToMapOnSD}{$OtherSD} = undef;
}
close M;
# Read in position to region ID
print "Read in position to region ID\n";
$FileHandle = new IO::Zlib;
if ($FileHandle->open($PositionToRegionFP, "rb")){
while (<$FileHandle>){
$_ =~ s/\n//g;
(my $Coord, my $RegionName) = split(m/\t/, $_);
(my $Chrom, my $Position) = split(m/~/, $Coord);
$PosToRegionName{$Chrom}{$Position} = $RegionName;
}
}
# Read in strands
print "Read in strands\n";
open S, "$RegionToStrandsFP" or die ("Can't open $RegionToStrandsFP\n");
while(<S>){
$_ =~ s/\n//g;
(my $RegionID, my $Strand) = split(m/\t/, $_);
$Strands{$RegionID} = $Strand;
}
close S;
# Tabulate variants #
print "Tabulate variants\n";
open O, ">$VariantsOutFP" or die ("Can't open $VariantsOutFP\n");
foreach my $VCF (@VCFs){
my $Caller = "";
my $Masking = "";
my $Analysis = "";
my $VARIANT_CALLER_SELECT = "";
$FileHandle = new IO::Zlib;
my $VCF_FilePath = $WORKING_DIR . "RAW/$SAMPLE_NAME/" . $VCF;
if ($VCF =~ m/\.gatk\./) {$Caller = "gatk";}
else {$Caller = "lofreq";}
if ($VCF =~ m/\.masked\./) {$Analysis = "MASKED";}
elsif ($VCF =~ m/\.ori\./) {$Analysis = "ORI";}
if ($Caller eq "lofreq") {$VARIANT_CALLER_SELECT = "LoFreq";}
elsif ($Caller eq "gatk") {$VARIANT_CALLER_SELECT = "GATK";}
if ($FileHandle->open($VCF_FilePath, "rb")){
while (<$FileHandle>){
next if ($_ =~ m/^#/);
my $Line = $_;
$Line =~ s/\n//g;
(my $VariantsRef, my $AFsRef, my $Depth, my $Qual) = DetermineVariantInVCF_Line ($Line, $VARIANT_CALLER_SELECT);
for (my $I = 0; $I < scalar @$VariantsRef; $I++){
my $Variant = $VariantsRef->[$I];
(my $Chrom, my $ChromStart, my $ChromEnd, undef) = split(m/~/, $Variant);
my $AF = $AFsRef->[$I];
$AF =~ s/AF\=//g;
my $Min = min($ChromStart, $ChromEnd);
my $Max = max($ChromStart, $ChromEnd);
my $BedStart = $Min-1;
my $VariantClass = "nonSD";
if (exists $SDsVAR{$Variant}){$VariantClass = "SD";}
next if ($AF eq "/");
$AF = sprintf("%.3f", $AF);
print O "$Chrom\t$BedStart\t$Max\t$Variant\t$SAMPLE_NAME\t$Depth\t$AF\t$Qual\t$Caller\t$Analysis\t$VariantClass\n";
if ($Caller eq "lofreq"){
$LoFreq_Variants{$Chrom}{$ChromStart}{$Variant}{$Analysis} = $AF;
}
if ($Caller eq "gatk"){
$GATK_Variants{$Chrom}{$ChromStart}{$Variant}{$Analysis} = $AF;
}
}
}
}
$FileHandle->close;
}
close O;
# Positions of interest #
print "Positions of interest\n";
foreach my $Chrom (keys %LoFreq_Variants){
foreach my $Start (keys %{$LoFreq_Variants{$Chrom}}){
foreach my $Variant (keys %{$LoFreq_Variants{$Chrom}{$Start}}){
$PosOfInterest{$Chrom}{$Start}{$Variant} = undef;
}
}
}
foreach my $Chrom (keys %GATK_Variants){
foreach my $Start (keys %{$GATK_Variants{$Chrom}}){
foreach my $Variant (keys %{$GATK_Variants{$Chrom}{$Start}}){
$PosOfInterest{$Chrom}{$Start}{$Variant} = undef;
}
}
}
foreach my $Chrom (keys %SDsBED){
foreach my $Start (keys %{$SDsBED{$Chrom}}){
foreach my $Variant (keys %{$SDsBED{$Chrom}{$Start}}){
$PosOfInterest{$Chrom}{$Start}{$Variant} = undef;
}
}
}
# Correct with coverage above threshold #
print "Correct with coverage above threshold\n";
$FileHandle = new IO::Zlib;
if ($FileHandle->open($CovFilePath, "rb")){
while (<$FileHandle>){
my $Line = $_;
$Line =~ s/\n//g;
(my $Chrom, my $Start, my $End, my $PosInInt, my $Cov) = split (m/\t/, $Line);
my $CovPos = $Start + $PosInInt;
next if (!exists $CoordinateMappings{"$Chrom\~$CovPos"});
if ($Cov >= 60){
$Coverage{$Chrom}{$CovPos} = $Cov;
if ( exists $LoFreq_Variants{$Chrom} &&
exists $LoFreq_Variants{$Chrom}{$CovPos}){
foreach my $Variant (keys %{$LoFreq_Variants{$Chrom}{$CovPos}}){
if (!exists $LoFreq_Variants{$Chrom}{$CovPos}{$Variant}{"masked"}){
$LoFreq_Variants{$Chrom}{$CovPos}{$Variant}{"masked"} = 0;
}
}
foreach my $Variant (keys %{$PosOfInterest{$Chrom}{$CovPos}}){
if (!exists $LoFreq_Variants{$Chrom}{$CovPos}{$Variant} &&
!exists $LoFreq_Variants{$Chrom}{$CovPos}{$Variant}{"masked"}){
$LoFreq_Variants{$Chrom}{$CovPos}{$Variant}{"masked"} = 0;
}
}
}
elsif ((!exists $LoFreq_Variants{$Chrom} ||
!exists $LoFreq_Variants{$Chrom}{$CovPos}) &&
exists $PosOfInterest{$Chrom} &&
exists $PosOfInterest{$Chrom}{$CovPos}){
foreach my $Variant (keys %{$PosOfInterest{$Chrom}{$CovPos}}){
$LoFreq_Variants{$Chrom}{$CovPos}{$Variant}{"masked"} = 0;
}
}
}
}
}
$FileHandle->close;
# Correct with coverage below threshold #
print "Correct with coverage below threshold\n";
foreach my $Chrom (sort keys %PosOfInterest){
foreach my $Start (sort keys %{$PosOfInterest{$Chrom}}){
next if (!exists $CoordinateMappings{"$Chrom\~$Start"});
if ( exists $LoFreq_Variants{$Chrom} &&
exists $LoFreq_Variants{$Chrom}{$Start}){
if (!exists $Coverage{$Chrom}{$Start}){
delete($LoFreq_Variants{$Chrom}{$Start});
foreach my $HomoloCoord (keys %{$CoordinateMappings{"$Chrom\~$Start"}}){
(my $HomChrom, my $HomPos) = split(m/~/, $HomoloCoord);
if ($LoFreq_Variants{$HomChrom}{$HomPos}){
delete($LoFreq_Variants{$HomChrom}{$HomPos});
}
}
}
}
}
}
open F1, ">$VariantsFilter1OutFP" or die ("can't open $VariantsFilter1OutFP\n");
open B, ">$VariantsFilter1BedFP" or die ("can't open $VariantsFilter1BedFP\n");
print F1 "Chromosome\tStart\tVAP\tVariantCall\tVAF_Masked\tMaskedCov\n";
foreach my $Chrom (sort keys %LoFreq_Variants){
foreach my $Pos (sort keys %{$LoFreq_Variants{$Chrom}}){
#print $Pos . "\n";
next if (!exists $Coverage{$Chrom}{$Pos});
foreach my $Variant (sort keys %{$LoFreq_Variants{$Chrom}{$Pos}}){
#print $Variant . "\n";
(my $ToMapOnChrom, my $ToMapOnStart, my $ToMapOnEnd, my $Change) = split (m/~/, $Variant);
(my $RefNucl, my $AltNucl) = split(m/\//, $Change);
if ($CoordinateMappings{"$Chrom\~$ToMapOnStart"} && $CoordinateMappings{"$Chrom\~$ToMapOnEnd"}){
if (! exists $MappedSDs{$Variant} &&
exists $LoFreq_Variants{$Chrom}{$Pos}{$Variant}{"MASKED"} &&
$LoFreq_Variants{$Chrom}{$Pos}{$Variant}{"MASKED"} >=0.15 &&
(! exists $GATK_Variants{$Chrom} ||
! exists $GATK_Variants{$Chrom}{$Pos} ||
! exists $GATK_Variants{$Chrom}{$Pos} ||
! exists $GATK_Variants{$Chrom}{$Pos}{$Variant} ||
! exists $GATK_Variants{$Chrom}{$Pos}{$Variant}{"ORI"})){
my $Present = 0;
my $Edge = 0;
### CHECK NEIGHBOURHOOD FOR INDELS ###
if ($RefNucl eq "-" || $AltNucl eq "-"){
for (my $I = $Pos-2; $I <= $Pos+2; $I++){
if (exists $GATK_Variants{$Chrom} &&
exists $GATK_Variants{$Chrom}{$I}){
foreach my $TryVariant (keys %{$GATK_Variants{$Chrom}{$I}}){
(undef, undef, undef, my $TryChange) = split(m/\~/, $TryVariant);
(my $TryRefNucl, my $TryAltNucl) = split(m/\//, $TryChange);
if (exists $GATK_Variants{$Chrom}{$I}{$TryVariant}{"ORI"} &&
($RefNucl eq "-" && $TryRefNucl eq "-") ||
($AltNucl eq "-" && $TryAltNucl eq "-")){
$Present = 1;
}
}
}
}
}
## CHECK IF ALL OTHERVARIANTS ARE ABSENT ###
my $OtherVariantsRef = ConstructOtherVariant($Variant, \%CoordinateMappings, \%PosToRegionName, \%Strands, \%SDsBED);
if ($Present == 0){
foreach my $OtherVariant (keys %$OtherVariantsRef){
(my $OtherChrom, my $OtherStart, my $OtherEnd, my $OtherChange) = split(m/\~/, $OtherVariant);
if (exists $GATK_Variants{$OtherChrom} &&
exists $GATK_Variants{$OtherChrom}{$OtherStart} &&
exists $GATK_Variants{$OtherChrom}{$OtherStart}{$OtherVariant} &&
exists $GATK_Variants{$OtherChrom}{$OtherStart}{$OtherVariant}{"ORI"}){
$Present = 1;
}
if (!exists $PosToRegionName{$OtherChrom} ||
!exists $PosToRegionName{$OtherChrom}{$OtherStart}){
$Edge = 1;
}
if ($Present+$Edge == 0 && ($RefNucl eq "-" || $AltNucl eq "-")){
for (my $I = $OtherStart-2; $I <= $OtherStart+2; $I++){
if (exists $GATK_Variants{$OtherChrom} &&
exists $GATK_Variants{$OtherChrom}{$I} &&
exists $GATK_Variants{$OtherChrom}{$I}){
foreach my $TryVariant (keys %{$GATK_Variants{$OtherChrom}{$I}}){
(undef, undef, undef, my $TryChange) = split(m/\~/, $TryVariant);
(my $TryRefNucl, my $TryAltNucl) = split(m/\//, $TryChange);
if (exists $GATK_Variants{$OtherChrom}{$I}{$TryVariant}{"ORI"} &&
($RefNucl eq "-" && $TryRefNucl eq "-") ||
($AltNucl eq "-" && $TryAltNucl eq "-")){
$Present = 1;
}
}
}
}
}
}
}
if ($Present+$Edge == 0){
print F1 $Chrom . "\t" . $Pos . "\t" . $Variant . "\t" . $Variant . "\t" . $LoFreq_Variants{$Chrom}{$Pos}{$Variant}{"MASKED"} . "\t" . $Coverage{$Chrom}{$Pos} . "\n";
(undef, my $StartPos, my $EndPos, undef) = split(m/\~/, $Variant);
my $BedStart = min($StartPos, $EndPos) - 1;
my $BedEnd = min($StartPos, $EndPos);
print B $Chrom . "\t" . $BedStart . "\t" . $BedEnd . "\t" . $Variant . "\n";
$F1Variants{$Variant} = "$Chrom\t$Pos\t$Variant\t$Variant\t$LoFreq_Variants{$Chrom}{$Pos}{$Variant}{\"MASKED\"}\t$Coverage{$Chrom}{$Pos}";
foreach my $OtherVariant (keys %$OtherVariantsRef){
(my $OtherChrom, my $OtherStart, my $OtherEnd, my $OtherChange) = split(m/\~/, $OtherVariant);
my $OtherRegionName = $PosToRegionName{$OtherChrom}{$OtherStart};
print F1 $OtherChrom . "\t" . $OtherStart . "\t" . $OtherVariant . "\t" . $Variant . "\t" . $LoFreq_Variants{$Chrom}{$Pos}{$Variant}{"MASKED"} . "\t" . $Coverage{$Chrom}{$Pos} . "\n";
my $OtherBedStart = min($OtherStart, $OtherEnd) - 1;
my $OtherBedEnd = min($OtherStart, $OtherEnd);
print B $OtherChrom . "\t" . $OtherBedStart . "\t" . $OtherBedEnd . "\t" . $OtherVariant . "\n";
$F1Variants{$OtherVariant} = "$OtherChrom\t$OtherStart\t$OtherVariant\t$Variant\t$LoFreq_Variants{$Chrom}{$Pos}{$Variant}{\"MASKED\"}\t$Coverage{$Chrom}{$Pos}";
}
}
}
}
}
}
}
close B;
close F1;
# Filter Out HP regions #
system("sort-bed $VariantsFilter1BedFP > $SortedVariantsFilter1BedFP");
system("bedmap --echo --echo-map --delim '\t' $SortedVariantsFilter1BedFP $HPsBedFP > $VariantsFilter1HpAnnoBedFP");
open HP, "$VariantsFilter1HpAnnoBedFP" or die ("Can't open $VariantsFilter1HpAnnoBedFP\n");
while(<HP>){
my $Line = $_;
$Line =~ s/\n//g;
my @LineValues = split(m/\t/, $Line);
my $Variation = $LineValues[3];
if (scalar @LineValues == 8){
$VarsToExclude{$Variation} = undef;
}
}
close HP;
# Trouble regions #
open S, "$TroubleSitesFP" or die ("Can't open $TroubleSitesFP\n");
while(<S>){
$_ =~ s/\n//g;
(my $Chrom, undef, my $Pos) = split(m/\t/, $_);
$SDtroublePos{$Chrom}{$Pos} = undef;
}
close S;
# Population frequency #
open P, "$CohortAF_FP" or die ("Can't open $CohortAF_FP");
while(<P>){