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CoNVaDING.pl
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#!/usr/bin/perl -w
use strict;
use warnings;
use diagnostics;
use File::Glob ':glob';
use File::Basename;
use Getopt::Long;
use List::Util qw(sum);
use Math::Complex;
use POSIX qw(ceil);
use POSIX qw(floor);
use Statistics::Normality 'shapiro_wilk_test';
print "\n#######################################\n";
print "COMMANDLINE OPTIONS IN AFFECT:\n";
foreach my $par (@ARGV){
print "$par\n";
}
print "#######################################\n";
######CHANGE VERSION PARAMETER IF VERSION IS UPDATED#####
my $version = "1.2.1";
##############################################################################################
##############################################################################################
## CoNVaDING, copy number variation detecting in next-generation sequencing gene panels ##
## Copyright (C) 2015 Freerk van Dijk & Lennart Johansson ##
## ##
## This file is part of CoNVaDING. ##
## ##
## CoNVaDING is free software: you can redistribute it and/or modify ##
## it under the terms of the GNU Lesser General Public License as published by ##
## the Free Software Foundation, either version 3 of the License, or ##
## (at your option) any later version. ##
## ##
## CoNVaDING is distributed in the hope that it will be useful, ##
## but WITHOUT ANY WARRANTY; without even the implied warranty of ##
## MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the ##
## GNU Lesser General Public License for more details. ##
## ##
## You should have received a copy of the GNU Lesser General Public License ##
## along with CoNVaDING. If not, see <http://www.gnu.org/licenses/>. ##
##############################################################################################
##############################################################################################
#Commandline variables
my ($help, $mode, $inputdir, $controlsdir, $outputdir, $bedfile, $rmdup, $sexchr, $sampleAsControl, $numBestMatchSamplesCmdL, $regionThreshold, $ratioCutOffLow , $ratioCutOffHigh, $zScoreCutOffLow, $zScoreCutOffHigh, $sampleRatioScore, $targetQcList, $percentageLessReliableTargets);
#### get options
GetOptions(
"h" => \$help,
"mode=s" => \$mode, #For options, see list below (default="StartWithBam")
"inputDir:s" => \$inputdir, #optional
"controlsDir:s" => \$controlsdir, #optional
"outputDir=s" => \$outputdir,
"bed:s" => \$bedfile, #optional
"controlSamples:s" => \$numBestMatchSamplesCmdL, #optional
"regionThreshold:s" => \$regionThreshold, #optional
"rmDup:s" => \$rmdup, #optional
"sexChr:s" => \$sexchr, #optional
"useSampleAsControl:s" => \$sampleAsControl, #optional
"ratioCutOffLow:s" => \$ratioCutOffLow, #optional
"ratioCutOffHigh:s" => \$ratioCutOffHigh, #optional
"zScoreCutOffLow:s" => \$zScoreCutOffLow, #optional
"zScoreCutOffHigh:s" => \$zScoreCutOffHigh, #optional
"sampleRatioScore:s" => \$sampleRatioScore, #optional
"targetQcList:s" => \$targetQcList, #optional
"percentageLessReliableTargets:s" => \$percentageLessReliableTargets #optional
);
usage() and exit(1) if $help;
#Obligatory args
usage() and exit(1) unless $mode;
usage() and exit(1) unless $outputdir;
#Add more parameters later
#Check input parameters
if (not defined $mode){ #If $mode is not defined, assign default value "StartWithBam"
$mode = "StartWithBam";
}
if ($mode eq "StartWithBam" || $mode eq "StartWithAvgCount" || $mode eq "StartWithMatchScore" || $mode eq "StartWithBestScore" || $mode eq "GenerateTargetQcList" || $mode eq "CreateFinalList") {
#continue
}else{ #Throw error
die "Mode $mode is not supported. Please read the manual.\n";
}
#If controlsdir is specified while running best score mode, throw warning
#Set default threshold values
if ($mode eq "StartWithBestScore"){
if (defined $controlsdir){
print "\n##### WARNING ##### WARNING #####\n";
print "User specified parameter \"controlsDir\" not used in best score analysis.";
print "\n##### WARNING ##### WARNING #####\n";
}
if (not defined $regionThreshold) {
$regionThreshold = 20;
}
if (not defined $ratioCutOffLow) {
$ratioCutOffLow = 0.65;
}
if (not defined $ratioCutOffHigh) {
$ratioCutOffHigh = 1.4;
}
if (not defined $zScoreCutOffLow) {
$zScoreCutOffLow = -3;
}
if (not defined $zScoreCutOffHigh) {
$zScoreCutOffHigh = 3;
}
}
#Set default threshold values when running in GenerateTargetQcList mode
if ($mode eq "GenerateTargetQcList"){
if (not defined $controlsdir) { #Throw error when controlsdir is not specified
die "Directory for controlsamples (-controlsDir) is not specified, please specify to continue analysis.\n";
}
if (not defined $regionThreshold) {
$regionThreshold = 20;
}
if (not defined $ratioCutOffLow) {
$ratioCutOffLow = 0.65;
}
if (not defined $ratioCutOffHigh) {
$ratioCutOffHigh = 1.4;
}
if (not defined $zScoreCutOffLow) {
$zScoreCutOffLow = -3;
}
if (not defined $zScoreCutOffHigh) {
$zScoreCutOffHigh = 3;
}
if (not defined $sampleRatioScore) {
$sampleRatioScore = 0.09;
}
#Set inputdir to same value as controlsdir
$inputdir = $controlsdir;
}
#Check if input directory exists, otherwise error and die
if ($mode eq "StartWithBam" || $mode eq "StartWithAvgCount" || $mode eq "StartWithMatchScore" || $mode eq "StartWithBestScore" || $mode eq "CreateFinalList"){
if (not defined $inputdir) { #Throw error when inputdir is not specified
die "Directory for input samples (-inputDir) is not specified, please specify to continue analysis.\n";
}
}
#Check if output directory exists, otherwise create it
if (!-d $outputdir) { #Output directory does not exist, create it
`mkdir -p $outputdir`;
}
my @bedfile;
#Check if BED file is specified for StartWithBam and StartWithAvgCount, else die and throw error message
if ($mode eq "StartWithBam" || $mode eq "StartWithAvgCount"){
if (not defined $bedfile){
die "Required BED file not specified, please specify to continue analysis.\n";
}
}elsif ($mode eq "StartWithMatchScore" || $mode eq "StartWithBestScore"){ #Throw warning saying BED file is not used in this analysis.
if (defined $bedfile){
print "\n##### WARNING ##### WARNING #####\n";
print "User specified parameter \"bed\" not used in this analysis.";
print "\n##### WARNING ##### WARNING #####\n";
}
}
#Check if number of control samples to use for best match is defined, else use default value
if (not defined $numBestMatchSamplesCmdL){
$numBestMatchSamplesCmdL = 30; #Default value for number of control samples to use
}
#Check if controlsdir exists for four param options
#if ($mode eq "StartWithBam" || $mode eq "StartWithAvgCount" || $mode eq "StartWithMatchScore" || $mode eq "GenerateTargetQcList"){ #Check if controlsdir is specified
if ($mode eq "StartWithBam" || $mode eq "StartWithAvgCount" || $mode eq "StartWithMatchScore"){ #Check if controlsdir is specified
if (not defined $controlsdir) { #Throw error when controlsdir is not specified
die "Directory for controlsamples (-controlsDir) is not specified, please specify to continue analysis.\n";
}else{
#Check if controls directory exists, otherwise create it
if (!-d $controlsdir) { #Controls directory does not exist, create it
`mkdir -p $controlsdir`;
}
}
#Check if controldir contains avg count files when useSampleAsControl paramater is not specified
if (not defined $sampleAsControl){
opendir(DIRHANDLE, $controlsdir) || die "Couldn't open directory $controlsdir: $!\n";
my @txt = grep { /\.normalized.coverage.txt$/ && -f "$controlsdir/$_" } readdir DIRHANDLE;
closedir DIRHANDLE;
if (@txt) { #not empty
#continue
my $numFiles = scalar(@txt); #If match score or best score mode is used, check if number of control sample files is greater than or equal to what user requested via parameter.
if ($mode eq "StartWithMatchScore") {
if ( $numFiles < $numBestMatchSamplesCmdL){
die "The number of controlsamples in $controlsdir is less than specified number of $numBestMatchSamplesCmdL controlsamples by \"controlSamples\" parameter.\n";
}
}
}else{
die "Directory specified by \"controlsDir\" parameter does not contain normalized coverage files.\n";
}
}
}
#Checks when running in CreateFinalList mode
if ($mode eq "CreateFinalList"){ #Check if targetQcList is specified
if (not defined $targetQcList) { #Throw error when targetQcList is not specified
die "Target QC list (-targetQcList) is not specified, please specify to continue analysis.\n";
}
if (not defined $percentageLessReliableTargets) {
$percentageLessReliableTargets = 20;
}
}
#Global vars
my $extension;
my $rmdupfile;
my $file;
my $filename;
my $bam;
my $chr;
my $start;
my $stop;
my $gene;
my $regioncov;
my $line;
my $covchrall;
my $covchrauto;
my $key;
my $covchrautosum;
my $covchrsexsum;
my $covchrautoval;
my $outputfile;
my $outputToWrite;
my $columnName;
my $mean;
my $sd;
my $fwdbam;
my $rvrsbam;
my $outputExtnsn;
my $colsToExtract;
my $perfectMatch=0;
my $TAi;
my $autoMean;
my $autoSD;
my $autoRatio;
my $autoZscore;
my $autoVc;
my $outputdirOriginal;
#Global arrays
my @inputfiles;
my @bamstocount;
my @filestodel;
my @covchrauto;
my @covchrsex;
my @genes;
my @headerarray;
my @indices;
my @values;
my @normCountFiles;
my @fwdControlLineArray;
my @rvrsControlLineArray;
my @fwds;
my @revrs;
my @sampleRatio;
my @TNciArray;
my @passSampleRatioSamples;
#Global hashes
my %coverage;
my %genehash;
my %counts;
my %autodiff;
my %sexdiff;
my %DFfile;
my %resultAuditTtest;
#Retrieve and print starttime
my $starttime = localtime();
print "\nStarting analysis $starttime\n";
##################################################################
##################################################################
#Start analysis from BAM file
if ($mode eq "StartWithBam"){
#continue
if (defined $rmdup) { #Remove duplicate switch added in cmdline
print "\n############\nrmdup switch detected, duplicate removal included in analysis\n############\n\n";
print "Starting removing duplicates and creating new BAM files..\n";
}
#Read BAM files
print "Reading BAM files to process..\n";
#Set file extension
$extension = ".bam";
readFile($inputdir, $extension);
#Start analysis from BAM file
startWithBam(\@inputfiles);
##################################################################
##################################################################
#Start analysis from average count files
}elsif ($mode eq "StartWithAvgCount"){
#Read count TXT files
print "Reading count files..\n";
$extension = ".txt";
readFile($inputdir, $extension);
print "Starting counts analysis..\n";
startWithAvgCount(\@inputfiles);
##################################################################
##################################################################
#Start analysis from match score
}elsif ($mode eq "StartWithMatchScore"){
#continue
if (defined $sexchr) { #Use sex chromosomes switch added in cmdline
print "\n\n############\nsexchr switch detected, sex chromosomes included in analysis\n############\n\n";
}
#Read count TXT files
print "Starting search for best match scores..\n";
print "Reading count files..\n";
$extension = ".normalized.coverage.txt";
readFile($controlsdir, $extension); #Read files in controls directory
my @controlfiles = @inputfiles;
undeff(@inputfiles);
readFile($inputdir, $extension); #Read files in input directory
print "Starting match score analysis..\n";
#Start analysis from match score file
startWithMatchScore($extension, \@inputfiles, \@controlfiles);
##################################################################
##################################################################
#Start analysis from best score
}elsif ($mode eq "StartWithBestScore"){
#continue
if (defined $sexchr) { #Use sex chromosomes switch added in cmdline
print "\n\n############\nsexchr switch detected, sex chromosomes included in analysis\n############\n\n";
}
#Read count TXT files
print "Starting search for best scores..\n";
#Read all normalized autosomal coverage control files into array
$extension = ".normalized.autosomal.coverage.all.controls.txt";
readFile($inputdir, $extension); #Read files in input directory
my @normAutoControls = @inputfiles;
undeff(@inputfiles);
#Read best match score files
print "Reading best match score files..\n";
$extension = ".best.match.score.txt";
readFile($inputdir, $extension); #Read files in input directory
print "Starting best score analysis..\n";
#Start CNV detection analysis
startWithBestScore(\@inputfiles, \@normAutoControls);
#empty output variable
undeff($outputToWrite);
##################################################################
##################################################################
#Generate target QC list from all controlsamples
}elsif ($mode eq "GenerateTargetQcList"){
#Read count TXT files
print "Reading controls directory..\n";
#Read all *.txt files in control directory into array
$extension = ".normalized.coverage.txt";
readFile($controlsdir, $extension); #Read files in controls directory
my @controlfiles = @inputfiles;
undeff(@inputfiles);
readFile($inputdir, $extension); #Read files in input directory
#Set temporary outputdir to do intermediate work
$outputdirOriginal = $outputdir;
$outputdir="$outputdirOriginal/tmpStartWithMatchScore/";
`mkdir $outputdir`;
#Start analysis from match score file
startWithMatchScore($extension, \@inputfiles, \@controlfiles);
#Read count TXT files
print "Starting search for best scores..\n";
#Read all normalized autosomal coverage control files into array
$extension = ".normalized.autosomal.coverage.all.controls.txt";
readFile("$outputdirOriginal/tmpStartWithMatchScore/", $extension); #Read files in input directory
my @normAutoControls = @inputfiles;
undeff(@inputfiles);
#Read best match score files
print "Reading best match score files..\n";
$extension = ".best.match.score.txt";
readFile("$outputdirOriginal/tmpStartWithMatchScore/", $extension); #Read files in input directory
print "Starting best score analysis..\n";
#Set temporary outputdir to do intermediate work
$outputdir="$outputdirOriginal/tmpStartWithBestScore/";
`mkdir $outputdir`;
$inputdir="$outputdirOriginal/tmpStartWithMatchScore/";
#Start CNV detection analysis
startWithBestScore(\@inputfiles, \@normAutoControls);
#empty output variable
undeff($outputToWrite);
#Remove temporary matchscore directory
`rm -r $outputdirOriginal/tmpStartWithMatchScore/`;
#Read all *.log files in control directory into array
$extension = ".log";
$inputdir = "$outputdirOriginal/tmpStartWithBestScore/";
readFile($inputdir, $extension); #Read files in controls directory
my @logfiles = @inputfiles;
undeff(@inputfiles);
#Extract sampleratio from *.log file
print "\n#######################################\n";
print "\n\nSamples failing sample CV threshold of $sampleRatioScore:\n\n";
foreach my $logfile (@logfiles){
my $grep = `grep SAMPLE_CV: $inputdir/$logfile`;
chomp $grep;
if ($grep =~ m/SAMPLE_CV: ([0-9].+)/gs){ #Check if sampleratio is below threshold(default 0.09), otherwise don't use it in targetlist analysis
my $sampleRatio = $1;
if ($sampleRatio <= $sampleRatioScore) {
$logfile =~ s/.log/.totallist.txt/gs; #Change *.log extension to *.totallist.txt
push(@passSampleRatioSamples, "$inputdir/$logfile");
}else{
#Print samples failing sample CV score to stdout
print "$logfile\n";
}
}
}
print "\n\n#######################################\n\n";
print "\nGenerating target QC list..\n";
#Calculate target autoVC
generateTargetQcList(\@passSampleRatioSamples);
print "\nDone generating target QC list\n";
##################################################################
##################################################################
#Create final list based on target QC file
}elsif ($mode eq "CreateFinalList"){ #Apply the target filtering using the file created in previous step
#Read shortlist TXT files
print "Reading input directory..\n";
#Read all *.txt files in input directory into array
$extension = ".shortlist.txt";
readFile($inputdir, $extension); #Read files in controls directory
#Generate the final list
createFinalList(\@inputfiles);
}
#Retrieve and print end time
my $endtime = localtime();
print "\nFinished analysis $endtime\n";
########################################################################################################
########## SUBS #################### SUBS #################### SUBS #################### SUBS ##########
########################################################################################################
###############################################
## Main code to generate final list using ##
## the target QC list ##
sub createFinalList{
my ($inputfiles) = @_;
#Read target QC list into array;
open(FILE, "$targetQcList") or die("Unable to open file: $!"); #Read count file
my @file= <FILE>;
close(FILE);
#Retrieve chr, start, stop and genename from file by searching column indices
my $header = uc($file[0]); #Header in uppercase
chomp $header;
#Extract columns from header and use as index
my @colNames = qw(CHR START STOP GENE);
getColumnIdx($header, \@colNames);
my $chrIdx = $indices[0];
my $startIdx = $indices[1];
my $stopIdx = $indices[2];
my $geneIdx = $indices[3];
my $lastFileIdx=$#file;
#Push every index into hash, to exclude from analysis later
my %toExclude;
foreach my $index (@indices){
$toExclude{ $index } = $index;
}
my @indicesCalculation;
#Iterate over header to extract indices to calculate 20% quality over
my @Header = split("\t", $header);
my $lastHeaderIdx = $#Header;
for (my $j=0; $j <= $lastHeaderIdx; $j++){
if (exists $toExclude{ $j }){
#Exclude from calculation
}else{
push(@indicesCalculation, $j);
}
}
my %targetQC;
#Retrieve chr, start, stop, gene for each line in target QC list
for (my $i=1; $i<=$lastFileIdx; $i++){ #Iterate over lines in file
$line=$file[$i];
chomp $line;
my @lines=split("\t",$line);
my $chr = $lines[$chrIdx];
my $start = $lines[$startIdx];
my $stop = $lines[$stopIdx];
my $gene = $lines[$geneIdx];
#Calculate number of samples passing cutoff, setting target to PASS or FAIL
my $key = "$chr\t$start\t$stop\t$gene";
my $total=0;
my $highQual=0;
my $lowQual=0;
foreach my $element (@indicesCalculation){
my $autoVC = $lines[$element];
$total++;
if ($autoVC eq "NA"){
$lowQual++;
}elsif ($autoVC <= 0.10) { #Good quality
$highQual++;
}else{ #Low quality target
$lowQual++;
}
}
my $perc = (($lowQual/$total)*100); #percentage low quality targets
if ($perc > $percentageLessReliableTargets){ #More than X percentage (default 20%) of targets are low quality, so FAIL
$targetQC{ $key } = "FAIL";
}else{
$targetQC{ $key } = "PASS";
}
}
#Iterate over input files, asses if failing targets are within the calls in the shortlist file
foreach my $shortlist (@$inputfiles){
print "\n\n#######################################\n";
print "Analyzing sample: $shortlist..\n";
#Read file into array;
my $dir;
my $ext;
($file,$dir,$ext) = fileparse($shortlist, qr/\.[^.]*/);
$file =~ s/.shortlist//gs;
open(FILE, "$inputdir/$shortlist") or die("Unable to open file: $!"); #Read count file
my @file= <FILE>;
close(FILE);
#Retrieve chr, start, stop, genename and region coverage from file by searching column indices
my $header = uc($file[0]); #Header in uppercase
chomp $header;
$outputToWrite .= "$header\n";
my @colNames = qw(CHR START STOP GENE);
getColumnIdx($header, \@colNames);
my $chrIdx = $indices[0];
my $startIdx = $indices[1];
my $stopIdx = $indices[2];
my $geneIdx = $indices[3];
my $lastFileIdx=$#file;
#Retrieve chr, start, stop, gene and regcov for each line in avg count file
for (my $k=1; $k<=$lastFileIdx; $k++){
$line=$file[$k];
chomp $line;
my @lines=split("\t",$line);
$chr=$lines[$chrIdx];
$chr =~ s/X/23/g;
$chr =~ s/Y/24/g;
$chr =~ s/Z/25/g;
$start=$lines[$startIdx];
$stop=$lines[$stopIdx];
$gene=$lines[$geneIdx];
my $totalTargets=0;
my $targetsFail=0;
#Check if all targets within a single event fail, if true filter event out of finallist.txt
foreach my $target (sort keys %targetQC){
my @targets = split("\t", $target);
my $chrQC = $targets[0];
$chrQC =~ s/X/23/g;
$chrQC =~ s/Y/24/g;
$chrQC =~ s/Z/25/g;
my $startQC = $targets[1];
my $stopQC = $targets[2];
my $geneQC = $targets[3];
if ($chrQC == $chr && $startQC >= $start && $stopQC <= $stop) { #chromosomes matching and start and stop of targetQC falls within the region extracted from *.shortlist.txt file
$totalTargets++;
#Check if target is pass or fail
my $check = $targetQC{ $target };
if ($check eq "FAIL") { #Target failed QC, count it
$targetsFail++;
}
}
}
#Check if number of failing targets equals number of total targets within this event, if true event fails quality threshold and is removed from *.finallist.txt
if ($totalTargets == $targetsFail) {
#event fails QC, don't write it to output
print "\nEvent failing target QC: $line\n";
}else{
$outputToWrite .= "$line\n";
}
}
#Write output to *.finallist.txt file
$outputfile = "$outputdir/$file.finallist.txt"; #Output filename
writeOutput($outputfile, $outputToWrite); #Write output to above specified file
print "#######################################\n\n";
undeff($outputToWrite);
}
}
###############################################
## Main code to generate target QC list from ##
## files in controls directory ##
sub generateTargetQcList{
my ($passSampleRatioSamples) = @_;
#Set header for output file
$outputToWrite = "CHR\tSTART\tSTOP\tGENE"; #Instantiate header to write
my %autoVcHash;
my @keyFiles;
my $lastInputfilesIdx = $#passSampleRatioSamples; #Idx of last inputfile
my @autoVCsToOutput;
for (my $j=0; $j <= $lastInputfilesIdx; $j++){ #Iterate over inputfiles
my $totallist = @$passSampleRatioSamples[$j];
#Read file into array;
my $dir;
my $ext;
($file,$dir,$ext) = fileparse($totallist, qr/\.[^.]*/);
#Append filename to outputToWrite to create header
$outputToWrite .= "\t$file";
open(FILE, "$totallist") or die("Unable to open file: $!"); #Read count file
my @file= <FILE>;
close(FILE);
#Retrieve chr, start, stop, genename and region coverage from file by searching column indices
my $header = uc($file[0]); #Header in uppercase
chomp $header;
#Extract columns from header and use as index
my @colNames = qw(CHR START STOP GENE AUTO_VC);
getColumnIdx($header, \@colNames);
my $chrIdx = $indices[0];
my $startIdx = $indices[1];
my $stopIdx = $indices[2];
my $geneIdx = $indices[3];
my $autoVcIdx = $indices[4];
my $lastFileIdx=$#file;
#Retrieve chr, start, stop, gene and regcov for each line in avg count file
my @autoVCs;
for (my $i=1; $i<=$lastFileIdx; $i++){ #Iterate over lines in file
$line=$file[$i];
chomp $line;
my @lines=split("\t",$line);
my $chr = $lines[$chrIdx];
my $start = $lines[$startIdx];
my $stop = $lines[$stopIdx];
my $gene = $lines[$geneIdx];
my $autoVc = $lines[$autoVcIdx];
if ($j == 0){ #If first file to pass, add chr,start,stop,gene to array, to use in output later
push(@autoVCsToOutput, "$chr\t$start\t$stop\t$gene");
}
push(@autoVCs, $autoVc); #Push autoVC in array
}
my $autoVcString = join("_", @autoVCs); #Join all autoVCs and push into string
$autoVcHash{ $file } = $autoVcString; #Push filename as key and string as value into hash
push(@keyFiles, $file); #Save filenames in particular order for output later
}
$outputToWrite .= "\n";
my $lastKeyFilesIdx = $#keyFiles; #Retrieve last idx from files
my @autoVcOutput;
#Loop through files to create matrix of autoVCs
for (my $i=0; $i <= $lastKeyFilesIdx; $i++){
my $key = $keyFiles[$i]; #Loop through filenames, take value from hash, split into array and loop through array per line
my @autoVCsToPrint = split("_", $autoVcHash{ $key }); #Push splitted value into array to iterate over later
my $lastAutoVCsToPrintIdx = $#autoVCsToPrint; #Last index from autoVC array
#Loop through all values and add them to original value in autoVCsToOutput array
for( my $k=0; $k<=$lastAutoVCsToPrintIdx; $k++){ #Iterate over all autoVC values for sample
my $autoVcToAppend = $autoVCsToPrint[$k]; #Retrieve autoVC for target k from sample
my $newElement;
if ($i == $lastKeyFilesIdx) { #last file to process, so add additional linebreak to value
$newElement = $autoVCsToOutput[$k] . "\t$autoVcToAppend\n"; #Take original value from array and append new autoVC for target k to it
}else{
$newElement = $autoVCsToOutput[$k] . "\t$autoVcToAppend";
}
#Splice output element k from array and update with new (above created) element
splice(@autoVCsToOutput, $k, 1, $newElement);
}
}
#Concat array with autoVCs to output string
my $outputString = join("", @autoVCsToOutput);
$outputToWrite .= $outputString;
#Write output to file
$outputfile = "$outputdirOriginal/targetQcList.txt"; #Output filename
writeOutput($outputfile, $outputToWrite); #Write output to above specified file
#Remove temporary directory
`rm -r $outputdir`;
}
###############################################
## Main code to extract region coverage from ##
## BAM file(s) ##
sub startWithBam{
my ($inputfiles) = @_;
foreach my $bam (@$inputfiles){
#Check if *.bam.bai or *.bai file exist, otherwise skip this bam file
my $dir;
my $ext;
($file,$dir,$ext) = fileparse($bam, qr/\.[^.]*/);
if (-e "$inputdir/$file.bai" || -e "$inputdir/$bam.bai") {
#Check if duplicates need to be removed
if (defined $rmdup){
#Process BAM files generating duplicate removed BAM files
rmDupBam("$bam");
print "Starting counts analysis..\n"; #Start to count regions
#rmdupfile is returned from rmDupBam function
countFromBam($rmdupfile);
print "\n";
rmTmpBAMs($outputdir, $filename); #Remove tmp generate files while running remove duplicates
}else{ #BAM files are already rmdupped, add them to list of files to process (retrieve them from inputdir cmdline)
print "Starting counts analysis..\n";
countFromBam("$inputdir/$bam");
print "\n";
}
print "Writing normalized coverage counts to: $outputfile\n\n\n";
undeff($outputToWrite);
#If $sampleAsControl variable is specified, write coverage.txt files to controlsdir too
#if (defined $sampleAsControl){
# `cp $outputfile $controlsdir`;
#}
}else{ #Don't process this file, because it doesn't have an indexfile
print "##### WARNING ##### WARNING #####\nCannot find an index file for file: $inputdir/$bam, skipping this file from analysis\n##### WARNING ##### WARNING #####\n\n";
}
}
}
sub startWithAvgCount{
my ($inputfiles) = @_;
foreach my $txt (@$inputfiles){
#Set header for output file
$outputToWrite = "CHR\tSTART\tSTOP\tGENE\tREGION_COV\tAVG_AUTOSOMAL_COV\tAVG_TOTAL_COV\tAVG_GENE_COV\tNORMALIZED_AUTOSOMAL\tNORMALIZED_TOTAL\tNORMALIZED_GENE\n";
#Read file into array;
my $dir;
my $ext;
($file,$dir,$ext) = fileparse($txt, qr/\.[^.]*/);
open(FILE, "$inputdir/$txt") or die("Unable to open file: $!"); #Read count file
my @file= <FILE>;
close(FILE);
#Retrieve chr, start, stop, genename and region coverage from file by searching column indices
my $header = uc($file[0]); #Header in uppercase
chomp $header;
my @colNames = qw(CHR START STOP GENE REGION_COV);
getColumnIdx($header, \@colNames);
my $chrIdx = $indices[0];
my $startIdx = $indices[1];
my $stopIdx = $indices[2];
my $geneIdx = $indices[3];
my $regcovIdx = $indices[4];
my $lastFileIdx=$#file;
#Retrieve chr, start, stop, gene and regcov for each line in avg count file
for (my $i=1; $i<=$lastFileIdx; $i++){
$line=$file[$i];
chomp $line;
my @lines=split("\t",$line);
my $chr=$lines[$chrIdx];
my $start=$lines[$startIdx];
my $stop=$lines[$stopIdx];
my $gene=$lines[$geneIdx];
my $regcov=$lines[$regcovIdx];
$key = $line;
#Calculate coverage from regions
calcGeneCov($chr, $start, $stop, $gene, $regcov, $line);
}
#Calculate coverage including sex chromomsomes
calcCovAutoSex(\@genes, \@covchrauto, \@covchrsex);
#Foreach line in input file write away all calculated stats/values
for (my $i=1; $i<=$lastFileIdx; $i++){
$line=$file[$i];
chomp $line;
my @lines=split("\t",$line);
my $chr=$lines[$chrIdx];
my $start=$lines[$startIdx];
my $stop=$lines[$stopIdx];
my $gene=$lines[$geneIdx];
my $regcov=$lines[$regcovIdx];
$key = $line;
#Write all values
$line = "$chr\t$start\t$stop\t$gene\t$regcov"; #Produce line to print in correct order for output
writeCountFile($line, $key, $gene, $covchrautoval, $covchrall, \%genehash, \%counts, \%coverage);
$covchrautosum = 0;
$covchrsexsum = 0;
}
$outputfile = "$outputdir/$file.normalized.coverage.txt"; #Output filename
writeOutput($outputfile, $outputToWrite, $controlsdir); #Write output to above specified file
#If $sampleAsControl variable is specified, write coverage.txt files to controlsdir too
#if (defined $sampleAsControl){
# `cp $outputfile $controlsdir`;
#}
#Empty all arrays and hashes
undef(@genes); undef(@covchrauto); undef(@covchrsex);
undef(%counts);
undef(%coverage);
undef(%genehash);
print "Finished processing file: $txt\n\n\n";
}
}
###############################################
## Main code to select most informative con- ##
## trol samples ##
sub startWithMatchScore{
my $extension = shift;
my ($inputfiles) = $_[0];
my ($controlfiles) = $_[1];
foreach my $inputfile (@$inputfiles){ #Open sample file
$outputExtnsn = "normalized.autosomal.coverage.all.controls.txt"; #Specify extension for output normalized coverage file
$colsToExtract = "CHR START STOP GENE REGION_COV NORMALIZED_AUTOSOMAL NORMALIZED_TOTAL"; #Specify Columns to extract
createNormalizedCoverageFiles($inputfile, $extension, $outputExtnsn, $colsToExtract, \@$controlfiles); #Give input file to analyze, extension, columns to extract and list of controlfiles to use to function to retrieve normalized coverage
#Open output bestmatch file
my $outputPostfixRemoved = $inputfile;
$outputPostfixRemoved =~ s/$extension//g; #Remove old extension from inputfile
$outputfile = "$outputdir/$outputPostfixRemoved.best.match.score.txt"; #Output filename
#Do additional checks to detect perfect match samples and check when this occurs if there still are enough samples available in controlsdirectory to continues analysis
my $numBestMatchSamplesToProcess = keys %autodiff; #Count number of samples to process (all - perfectMatch)
my $numBestMatchSamples=$numBestMatchSamplesCmdL;
if ( $perfectMatch > 0){
if ($numBestMatchSamplesToProcess == $numBestMatchSamples){ #Detected perfect matches, but number of samples to process is the same as number of samples requested on cmdline
print "\n##### WARNING ##### WARNING #####\n";
print "Detected $perfectMatch perfect match(es) between sample and controlsamples, excluding these samples from the analysis.";
print "\n##### WARNING ##### WARNING #####\n";
}elsif ($numBestMatchSamplesToProcess >= ($numBestMatchSamples+$perfectMatch)){ #If there are more or equal #samples in controlsdir then specified on cmdline, use best #cmdline samples
$numBestMatchSamples=$numBestMatchSamples;
}else{ #Less samples than cmdline specified available, continue analysis but throw warning
print "\n##### WARNING ##### WARNING #####\n";
print "Detected $perfectMatch perfect match(es) between sample and controlsamples, which means only $numBestMatchSamplesToProcess instead of $numBestMatchSamples samples from controls directory are used for Match score analysis.";
print "\n##### WARNING ##### WARNING #####\n";
$numBestMatchSamples = $numBestMatchSamplesToProcess;
}
}
$perfectMatch=0; #Reset perfectMatch variable
print "\n#######################################\n";
print "Selecting best $numBestMatchSamples control samples for analysis..\n";
print "#######################################\n";
my @keys;
my @vals;
if (defined $sexchr) { #Use sex chromosomes switch, print all abs diffs including sex chromosomes
@keys = sort { $sexdiff{$a} <=> $sexdiff{$b} } keys(%sexdiff);
@vals = @sexdiff{@keys};
}else { #Only use autosomal chrs
@keys = sort { $autodiff{$a} <=> $autodiff{$b} } keys(%autodiff);
@vals = @autodiff{@keys};
}
undeff($outputToWrite);
$outputToWrite= "SAMPLE\tSAMPLE_PATH\tCONTROL_SAMPLE\tCONTROL_SAMPLE_PATH\tAVERAGE_BEST_MATCH_SCORE\n"; #Assign header to output best match file
for (my $k=0; $k < $numBestMatchSamples; $k++){
print "Control: " . $keys[$k] . "\t\t\tAvg abs diff: " . $vals[$k] . "\n";
my $lin = $inputfile . "\t$inputdir/$inputfile\t" . $keys[$k] . "\t$controlsdir/" . $keys[$k] . "\t" . $vals[$k] . "\n";
$outputToWrite .= $lin; #concatenate full generated line to files
}
print "#######################################\n\n";
writeOutput($outputfile, $outputToWrite); #Write output to above specified file
undef(%autodiff);
undef(%sexdiff);
}
}
###############################################
## Main code to detect CNVs ##
sub startWithBestScore{
my ($inputfiles) = $_[0];
my ($normAutoControls) = $_[1];
my $lastFileIdx = $#inputfiles;
for (my $m=0; $m<=$lastFileIdx; $m++){
my $inputfile = $inputfiles[$m];
my @sampleFile;
my @controlFile;
my @controlChr;
my @controlStart;
my @controlStop;
my @controlGene;
print "\nAnalyzing sample: $inputfile..\n";
$outputToWrite= "CHR\tSTART\tSTOP\tGENE\t$inputfile\t"; #Assign header to output best match file
open(INPUTFILE, "$inputdir/$inputfile") or die("Unable to open file: $!"); #Read best match file
my @inputfile= <INPUTFILE>;
close(INPUTFILE);
my $header = uc($inputfile[0]); #Header in uppercase
chomp $header;
my @colNames = qw(SAMPLE SAMPLE_PATH CONTROL_SAMPLE CONTROL_SAMPLE_PATH);
getColumnIdx($header, \@colNames);
my $sampleIdx = $indices[0];
my $samplePathIdx = $indices[1];
my $controlIdx = $indices[2];
my $controlPathIdx = $indices[3];
my @array = split("\t", $header);
my $samplename = $array[$sampleIdx];
my $samplefile = $array[$samplePathIdx];
my $controlname = $array[$controlIdx];
my $controlfile = $array[$controlPathIdx];
my $lastLine=$#inputfile;
#Initialize arrays for arrayreferences
my @NORMAUTOSOMAL;
my @NORMTOTAL;
my @NORMGENE;
# Read samplefile into array (only do this once)
my $currentline = $inputfile[1]; #Open first samplefile from inputfile
my @crntlnArray = split("\t", $currentline);
# Read input samplefile into array
open(SAMPLEFILE, "$crntlnArray[$samplePathIdx]") or die("Unable to open file: $!"); #Read SAMPLE best match file
@sampleFile= <SAMPLEFILE>;
close(SAMPLEFILE);
my $headerSample = uc($sampleFile[0]); #Header in uppercase
chomp $headerSample;
# Read normalized_autosomal values file
#Open norm auto file, further on calculate if sample is within 3SD from mean, otherwise exclude in sampleratio calculation
my @normAutoCon = @$normAutoControls;
my $normautofile = $normAutoCon[$m];
open(NORMAUTOFILE, "$inputdir/$normautofile") or die("Unable to open file: $!"); #Read count file
my @normautofile= <NORMAUTOFILE>;
close(NORMAUTOFILE);
#Extract sample file header indices
@colNames = qw(CHR START STOP GENE NORMALIZED_AUTOSOMAL NORMALIZED_TOTAL NORMALIZED_GENE);
getColumnIdx($headerSample, \@colNames);
my $chrIdxControl = $indices[0];
my $startIdxControl = $indices[1];
my $stopIdxControl = $indices[2];
my $geneIdxControl = $indices[3];
my $normAutoIdxSample = $indices[4];
my $normSexIdxSample = $indices[5];
my $normGeneIdxSample = $indices[6];
#Read through samplefile and push all three values into array of arrays;
my $lastLineSampleFile = $#sampleFile;
my @sampleNormAuto;
my @sampleNormTotal;
my @sampleNormGene;
for (my $i=1; $i<=$lastLineSampleFile; $i++){
my $currentline = $sampleFile[$i];
my @crntlnArray = split("\t", $currentline);
push(@controlChr, $crntlnArray[$chrIdxControl]);
push(@controlStart, $crntlnArray[$startIdxControl]);
push(@controlStop, $crntlnArray[$stopIdxControl]);
push(@controlGene, $crntlnArray[$geneIdxControl]);
push(@sampleNormAuto, $crntlnArray[$normAutoIdxSample]);
push(@sampleNormTotal, $crntlnArray[$normSexIdxSample]);
push(@sampleNormGene, $crntlnArray[$normGeneIdxSample]);
}
my $refNormAuto = \@sampleNormAuto; #Create array references
my $refNormTotal = \@sampleNormTotal;
my $refNormGene = \@sampleNormGene;
push(@NORMAUTOSOMAL, $refNormAuto);
push(@NORMTOTAL, $refNormTotal);
push(@NORMGENE, $refNormGene);
#Finalize header line for outputfile
my $lin = "AUTO_RATIO\tAUTO_ZSCORE\tAUTO_VC\tGENE_RATIO\tGENE_ZSCORE\tGENE_VC\tSHAPIRO-WILK\n";
$outputToWrite .= $lin; #concatenate full generated line to files
#Iterate over controlfiles and put into array of arrays
for (my $i=1; $i<=$lastLine; $i++){
# Read line from inputfile
my $currentline = $inputfile[$i];
my @crntlnArray = split("\t", $currentline);
#Read control file into array
open(CONTROLFILE, "$crntlnArray[$controlPathIdx]") or die("Unable to open file: $!"); #Read CONTROL best match file
@controlFile= <CONTROLFILE>;
close(CONTROLFILE);
#Control file is now in an array
my $headerControl = uc($controlFile[0]); #Header in uppercase
chomp $headerControl;
#Extract control header columnindices
my @colNames = qw(NORMALIZED_AUTOSOMAL NORMALIZED_TOTAL NORMALIZED_GENE);
getColumnIdx($headerControl, \@colNames);
my $normAutoIdxControl = $indices[0];
my $normSexIdxControl = $indices[1];
my $normGeneIdxControl = $indices[2];
my $lastLineControlFile = $#controlFile;
#Walk through contents of control file
my @controlNormAuto;
my @controlNormTotal;
my @controlNormGene;
for (my $i=1; $i<=$lastLineControlFile; $i++){
my $currentline = $controlFile[$i];
my @crntlnArray = split("\t", $currentline);
push(@controlNormAuto, $crntlnArray[$normAutoIdxControl]);
push(@controlNormTotal, $crntlnArray[$normSexIdxControl]);
push(@controlNormGene, $crntlnArray[$normGeneIdxControl]);
}
my $refNormAuto = \@controlNormAuto; #Create array references
my $refNormTotal = \@controlNormTotal;
my $refNormGene = \@controlNormGene;
push(@NORMAUTOSOMAL, $refNormAuto);