kantale/HLA-LA.pl

## HLA-LA.pl
#!/usr/bin/env perl

# ./inferHLATypes.pl --BAM /gpfs1/well/gsk_hla/bam_output/AA02O9Q_Z2.bam --graph PRG_MHC_GRCh38_withIMGT --sampleID NA12878Direct

use warnings;
use strict;
use FindBin;
use File::Spec;
use Getopt::Long;
use Data::Dumper;
use Sys::Hostname;
use Cwd qw/getcwd abs_path/;

$| = 1;
my $this_bin_dir = $FindBin::RealBin;

my $_BAM;
my $graph;
my $GRAPHDIR;
my $sampleID;
my $qsub;
my $samtools_bin;
my $bwa_bin;
my $java_bin;
my $picard_sam2fastq_bin;
my $workingDir_param;
my $maxThreads = 1;
my $moreReferencesDir;
my $extractExonkMerCounts;
my $longReads = 0;
my $testing = 0;
GetOptions (
	'BAM:s' => \$_BAM,
	'graph:s' => \$graph,
	'GRAPHDIR:s' => \$GRAPHDIR,
	'sampleID:s' => \$sampleID,
	'qsub:s' => \$qsub,
	'workingDir:s' => \$workingDir_param,
	'samtools_bin:s' => \$samtools_bin,
	'bwa_bin:s' => \$bwa_bin,
	'java_bin:s' => \$java_bin,
	'picard_sam2fastq_bin:s' => \$picard_sam2fastq_bin,
	'maxThreads:s' => \$maxThreads,
	'moreReferencesDir:s' => \$moreReferencesDir,
	'extractExonkMerCounts:s' => \$extractExonkMerCounts,
	'longReads:s' => \$longReads,
	'testing:s' => \$testing,
);

if ($extractExonkMerCounts)
{
	warn "--extractExonkMerCounts 1: This feature is experimental and unlikely to work on your machine";
	die unless(-e '../exonkMerExtraction/GRCh38.forkMers');
	die unless(-e '../exonkMerExtraction/exonCoordinates_manual.txt.forExtraction');
}

unless((not $longReads) or ($longReads eq 'ont2d') or ($longReads eq 'pacbio'))
{
	die "Please specify --longReads ont2d or --longReads pacbio";
}

my %paths_ini;
my $paths_ini = $this_bin_dir . '/paths.ini';
if(-e $paths_ini)
{
	open(INI, '<', $paths_ini) or die "Cannot open $paths_ini";
	while(<INI>)
	{
		chomp;
		next unless($_);
		$_ =~ s/[\n\r]//g;
		next if($_ =~ /^\s+$/);
		die "Invalid line format in $paths_ini -- expect all lines to be either empty or key=value pairs" unless($_ =~ /^(.+)=(.*)$/);
		my $id = $1;
		my @alts = split(/,/, $2);
		$paths_ini{$id} = \@alts;
	}
	close(INI);
}
$samtools_bin = find_path('samtools_bin', $samtools_bin, 'samtools');
$bwa_bin = find_path('bwa_bin', $bwa_bin, 'bwa');
$java_bin = find_path('java_bin', $java_bin, 'java');
$picard_sam2fastq_bin = find_path('picard_sam2fastq_bin', $picard_sam2fastq_bin, 'picard');

my $FASTQ_extraction_command_part1;
if($picard_sam2fastq_bin =~ /SamToFastq\.jar$/)
{
	$FASTQ_extraction_command_part1 = qq($java_bin -Xmx10g -XX:-UseGCOverheadLimit -jar $picard_sam2fastq_bin);
}
elsif($picard_sam2fastq_bin =~ /picard-tools$/)
{
	$FASTQ_extraction_command_part1 = qq($picard_sam2fastq_bin SamToFastq);
}
elsif($picard_sam2fastq_bin =~ /picard\.jar$/)
{
        $FASTQ_extraction_command_part1 = qq($java_bin -Xmx10g -XX:-UseGCOverheadLimit -jar $picard_sam2fastq_bin SamToFastq);
}
elsif($picard_sam2fastq_bin =~ /picard$/)
{
	$FASTQ_extraction_command_part1 = qq($picard_sam2fastq_bin SamToFastq);
}
else
{
	die "I can't interpret the specified Picard command: $picard_sam2fastq_bin";
}

if($testing)
{
	my $previous_dir = getcwd;
	chdir($this_bin_dir) or die "Cannot cd into $this_bin_dir";
	die "Binary ../bin/HLA-LA (from $this_bin_dir) not there!" unless(-e '../bin/HLA-LA');
	my $cmd_test = qq(../bin/HLA-LA	--action testBinary);
	system($cmd_test) and die "HLA*LA test command $cmd_test failed";
	chdir($previous_dir) or die "Cannot chdir into $previous_dir";

	print "HLA-LA.pl test\n\n";
	print "\t", "samtools_bin", ": ", $samtools_bin, "\n";
	print "\t", "bwa_bin", ": ", $bwa_bin, "\n";
	print "\t", "java_bin", ": ", $java_bin, "\n";
	print "\t", "picard_sam2fastq_bin", ": ", $picard_sam2fastq_bin, "\n";
	exit 0;
}


my $working_dir;
if($paths_ini{workingDir}[0] and not defined $workingDir_param)
{
	$working_dir = $paths_ini{workingDir}[0];
	$working_dir =~ s/\$HLA\-LA\-DIR/$this_bin_dir/;
}
else
{
	unless(defined $workingDir_param)
	{
		die "\n\nPlease specify a working directory via --workingDir.\n\nOutput for sample with ID \$sampleID will go a correspondingly named sub-directory of the working directory.\n\nFor example, if --workingDir is /path/working, and --sampleID is mySample, then the output will go into directory /path/working/mySample.\n\n";
	}
	$working_dir = $workingDir_param;
}

unless(-d $working_dir)
{
	die "\n\nSpecified working directory $working_dir is either not present or not a directory.\n\nYou might have specified an invalid path via --workingDir.\n\n";
}

$working_dir = abs_path($working_dir);

unless($sampleID =~ /^\w+$/)
{
	die "Please use only alphanumeric characters - \\w+ - for --sampleID";
}
my $working_dir_thisSample = $working_dir . '/' . $sampleID;


print "HLA-LA.pl\n\n";

print "Identified paths:\n";
print "\t", "samtools_bin", ": ", $samtools_bin, "\n";
print "\t", "bwa_bin", ": ", $bwa_bin, "\n";
print "\t", "java_bin", ": ", $java_bin, "\n";
print "\t", "picard_sam2fastq_bin", ": ", $picard_sam2fastq_bin, "\n";
print "\t", "General working directory", ": ", $working_dir, "\n";
print "\t", "Sample-specific working directory", ": ", $working_dir_thisSample, "\n";

print "\n";


unless(-d $working_dir_thisSample)
{
	mkdir($working_dir_thisSample) or die "Cannot mkdir $working_dir_thisSample";
}

my $samtools_version = `$samtools_bin --version` ;
die "Can't parse samtools version output" unless($samtools_version =~ /samtools ([\d\.]+)/);
$samtools_version = $1;
my $samtools_version_numeric = $samtools_version;
if($samtools_version_numeric =~ /^(\d+)\.(\d+)\.(\d+)$/)
{
	$samtools_version_numeric = $1 . '.' . $2 . $3;
}
#unless($samtools_version_numeric >= 1.3)
#{
#	die "I need samtools >=1.3";
#}


#my $BAM = File::Spec->abs2rel($_BAM);
my $BAM = $_BAM;
print "1343 BAM: $BAM \n";
unless(-e $BAM)
{
	die "BAM (or CRAM) $BAM (inferred from input $_BAM) not existing" unless(-e $BAM);
	unless((-e $BAM . '.bai') or (-e $BAM . '.crai'))
	{
		die "File $BAM does not appear to be indexed";
	}
}

my $full_graph_dir = $GRAPHDIR . '/' . $graph;
my $known_references_dir = $full_graph_dir . '/knownReferences';
unless(-e $full_graph_dir)
{
	die "Graph directory $full_graph_dir not found - valid graph names are subdirectories of the graphs directory in the HLA-LA root";
}
unless((-e $full_graph_dir . '/sequences.txt') and ((-e $full_graph_dir . '/extendedReferenceGenomePath.txt') or (-e $full_graph_dir . '/extendedReferenceGenome/extendedReferenceGenome.fa')) and (-d $known_references_dir))
{
	die "Graph directory $full_graph_dir does not seem to be complete - does this directory specify a valid graph for HLA-LA?";
}

# get index for this BAM
my $command_idxstats = qq($samtools_bin idxstats $BAM);
my $command_idxstats_output = `$command_idxstats`;
unless($command_idxstats_output)
{
	die "Didn't get a sensible output from command $command_idxstats";
}
my %BAM_idx;
my @BAM_idx_contigOrder;
my @idxstats_lines = split(/\n/, $command_idxstats_output);
foreach my $l (@idxstats_lines)
{
	my @l_fields = split(/\t/, $l);
	die unless(scalar(@l_fields) >= 1);
	die if(exists $BAM_idx{$l_fields[0]});
	$BAM_idx{$l_fields[0]} = $l_fields[1];
	push(@BAM_idx_contigOrder, $l_fields[0]);
}

my @files_references = glob($known_references_dir . '/*.txt');
die "No known reference files in knownReferences ($full_graph_dir)?" unless(@files_references);
my $additional_references_dir = $this_bin_dir . '/additionalReferences/' . $graph;
if(-e $additional_references_dir)
{
	my @additional_files_references = glob($additional_references_dir . '/*.txt');
	push(@files_references, @additional_files_references);
}
if($moreReferencesDir and (-e $moreReferencesDir))
{
	my $mD = $moreReferencesDir . '/'  . $graph;
	if(-d $mD)
	{
		my @even_more_files_references = glob($mD . '/*.txt');
		push(@files_references, @even_more_files_references);
	}
}

my @compatible_files;
my %extractContigs_complete_byFile;
my %extractContigs_partial_byFile;
foreach my $f (@files_references)
{
	open(F, '<', $f) or die "Cannot open $f";
	my $F_firstLine = <F>;
	chomp($F_firstLine);
	my @firstLine_fields = split(/\t/, $F_firstLine);
	my @expected_firstLine_fields = qw/contigID contigLength ExtractCompleteContig PartialExtraction_Start PartialExtraction_Stop/;
	die "Incorrect header for $f ($#firstLine_fields vs $#expected_firstLine_fields)" unless($#firstLine_fields == $#expected_firstLine_fields);
	for(my $i = 0; $i <= $#firstLine_fields; $i++)
	{
		die "Incorrect header for $f" unless($firstLine_fields[$i] eq $expected_firstLine_fields[$i]);
	}

	my $n_contigs = 0;
	my $n_contigs_matching = 0;
	while(<F>)
	{
		my $line = $_;
		chomp($line);
		next unless($line);
		my @line_fields = split(/\t/, $line, -1);
		$n_contigs++;
		if((exists $BAM_idx{$line_fields[0]}) and ($BAM_idx{$line_fields[0]} == $line_fields[1]))
		{
			$n_contigs_matching++;
		}
		else
		{
			# print "Mismatch $line_fields[0] - $line_fields[1] - " . $BAM_idx{$line_fields[0]} . "\n";
		}

		die if(($line_fields[2]) and (($line_fields[3]) and ($line_fields[4])));

		if($line_fields[2])
		{
			if($line_fields[0] eq '*')
			{
				$extractContigs_complete_byFile{$f}{$line_fields[0]} = 1;
			}
			else
			{
				$extractContigs_partial_byFile{$f}{$line_fields[0]} = [1, $line_fields[1]];
			}
		}

		if(($line_fields[3]) and ($line_fields[4]))
		{
			die if($line_fields[0] eq '*');
			die "Coordinate field $line_fields[3] has non-numeric characters" unless($line_fields[3] =~ /^\d+$/);
			die "Coordinate field $line_fields[4] has non-numeric characters" unless($line_fields[4] =~ /^\d+$/);

			$extractContigs_partial_byFile{$f}{$line_fields[0]} = [$line_fields[3], $line_fields[4]];
		}
	}
	close(F);

	if(($n_contigs_matching == $n_contigs) and ($n_contigs == scalar(@BAM_idx_contigOrder)))
	{
		push(@compatible_files, $f);
	}
}

if(scalar(@compatible_files) == 0)
{
	die "Have found no compatible reference specifications in $known_references_dir - create a file for this BAM file and try again.";
}
if(scalar(@compatible_files) > 1)
{
	die "Found more than one compatible reference file in $known_references_dir - a duplicate?\n\n".Dumper(\@compatible_files);
}

my $compatible_reference_file = $compatible_files[0];

my @refIDs_for_extraction;
foreach my $refID (@BAM_idx_contigOrder)
{
	next if($refID eq '*');
	die if((exists $extractContigs_complete_byFile{$compatible_reference_file}{$refID}) and (exists $extractContigs_partial_byFile{$compatible_reference_file}{$refID}));
	die unless((not defined $extractContigs_complete_byFile{$compatible_reference_file}{$refID}) or ($extractContigs_complete_byFile{$compatible_reference_file}{$refID} eq '0') or ($extractContigs_complete_byFile{$compatible_reference_file}{$refID} eq '1'));
	if($extractContigs_complete_byFile{$compatible_reference_file}{$refID})
	{
		push(@refIDs_for_extraction, $refID);
	}
	if($extractContigs_partial_byFile{$compatible_reference_file}{$refID})
	{
		push(@refIDs_for_extraction, $refID . ':' . $extractContigs_partial_byFile{$compatible_reference_file}{$refID}[0] . '-' . $extractContigs_partial_byFile{$compatible_reference_file}{$refID}[1]);
	}
}

die "No contigs for extraction specified in $compatible_reference_file?" unless(scalar(@refIDs_for_extraction));

my $target_extraction = $working_dir_thisSample . '/extraction.bam';


my $target_extraction_mapped = $working_dir_thisSample . '/extraction_mapped.bam';
my $extraction_command = qq($samtools_bin view -bo $target_extraction_mapped $BAM ).join(' ', @refIDs_for_extraction);
print "Extract reads from ", scalar(@refIDs_for_extraction), " regions...\n";
if(system($extraction_command) != 0)
{
	die "Extraction command $extraction_command failed";
}

if($extractContigs_complete_byFile{$compatible_reference_file}{'*'})
{
	my $target_extraction_unmapped = $working_dir_thisSample . '/extraction_unmapped.bam';

	my $extraction_command_unmapped = qq($samtools_bin view -b -f 4 $BAM > $target_extraction_unmapped);
	print "Extract unmapped reads...\n";
	print "extraction_command_unmapped=$extraction_command_unmapped\n";
	print "BAM: $BAM\n";

	if(system($extraction_command_unmapped) != 0)
	{
		die "Extraction command $extraction_command_unmapped failed";
	}

	unlink($target_extraction) if (-e $target_extraction);
	my $extraction_command_merge = qq($samtools_bin merge $target_extraction $target_extraction_mapped $target_extraction_unmapped);
	print "Merging...\n";
	if(system($extraction_command_merge) != 0)
	{
		die "Merge command $extraction_command_merge failed";
	}
}
else
{
	my $mv_command = qq(mv $target_extraction_mapped $target_extraction);
	if(system($mv_command) != 0)
	{
		die "Move command $mv_command failed";
	}
}

my $index_command = qq($samtools_bin index $target_extraction);
print "Indexing...\n";
if(system($index_command) != 0)
{
	die "Index command $index_command failed";
}

my $target_FASTQ_1 = $working_dir_thisSample . '/R_1.fastq';
my $target_FASTQ_2 = $working_dir_thisSample . '/R_2.fastq';
my $target_FASTQ_U = $working_dir_thisSample . '/R_U.fastq';
my $target_FASTQ_U_split = $working_dir_thisSample . '/R_U.fastq.splitLongReads';
my $FASTQ_extraction_command = qq($FASTQ_extraction_command_part1 VALIDATION_STRINGENCY=LENIENT I=$target_extraction F=$target_FASTQ_1 F2=$target_FASTQ_2 FU=$target_FASTQ_U 2>&1);

print "Extract FASTQ...\n\t$FASTQ_extraction_command\n";
my $FASTQ_extraction_output = `$FASTQ_extraction_command`;
#if(($FASTQ_extraction_output =~ /Exception/) or ($FASTQ_extraction_output !~ /net.sf.picard.sam.SamToFastq done/))
if(($FASTQ_extraction_output !~ /picard.sam.SamToFastq done/))
{
	die "Picard output: \n\n" . $FASTQ_extraction_output . "\n\nExtraction command $FASTQ_extraction_command $! \n\nAbort because the Picard FASTQ extraction process might have failed. I think so because I could not find the string 'picard.sam.SamToFastq done' in the Picard output.\n\n";
}

if($longReads)
{
	die "You activated --longReads, but the two files $target_FASTQ_1 and $target_FASTQ_2 (which store paired-end reads) are not empty - this is weird, and I will abort." unless(((-s $target_FASTQ_1) == 0) && ((-s $target_FASTQ_2) == 0));
	unless((-s $target_FASTQ_U) > 0)
	{
		die "You activated --longReads, but my attempt to extract long unpaired reads from the specified input BAM failed. Abort.";
	}

	open(INLONG, '<', $target_FASTQ_U) or die;
	open(OUTLONG, '>', $target_FASTQ_U_split) or die;
	while(<INLONG>)
	{
		chomp;
		my $readID = $_;
		die unless(substr($readID, 0, 1) eq '@');
		my $sequence = <INLONG>; chomp($sequence);
		my $plus = <INLONG>;
		my $qualities = <INLONG>; chomp($qualities);
		die unless(substr($plus, 0, 1) eq '+');
		die unless(length($sequence) == length($qualities));


		if(length($sequence) < 50000)
		{
			print OUTLONG $readID, "\n", $sequence, "\n", "+\n", $qualities, "\n";
		}
		else
		{
			my $runningI = 0;
			while(length($sequence))
			{
				die unless(length($sequence) == length($qualities));

				my $thisPart_readID = '>rP' .  $runningI . substr($readID, 1);

				my $extractionLength = (length($sequence) > 50000) ? 50000 : length($sequence);
				print OUTLONG $thisPart_readID, "\n", substr($sequence, 0, $extractionLength), "\n", "+\n", substr($qualities, 0, $extractionLength), "\n";

				substr($sequence, 0, $extractionLength) = '';
				substr($qualities, 0, $extractionLength) = '';

				$runningI++;
			}
		}
	}
	close(INLONG);
	close(OUTLONG);


}
else
{
	die "You didn't activate --longReads, but the two files $target_FASTQ_1 and $target_FASTQ_2 (which store paired-end reads) are empty - this is weird, and I will abort." unless(((-s $target_FASTQ_1) > 0) && ((-s $target_FASTQ_2) > 0));
}

#if(system($FASTQ_extraction_command) != 0)
#{
#}

my $mapAgainstCompleteGenome = ($extractContigs_complete_byFile{$compatible_reference_file}{'*'}) ? 1 : 0;
#$mapAgainstCompleteGenome = 0;

if($extractExonkMerCounts)
{
	die if($longReads);
	die unless(-e '../exonkMerExtraction/GRCh38.forkMers');
	die unless(-e '../exonkMerExtraction/exonCoordinates_manual.txt');

	my $command_extraction = qq(perl extractkMerCounts.pl --sampleID $sampleID --outputDirectory $working_dir_thisSample --referenceGenome ../exonkMerExtraction/GRCh38.forkMers --exonCoordinates ../exonkMerExtraction/exonCoordinates_manual.txt --FASTQ1 $target_FASTQ_1 --FASTQ2 $target_FASTQ_2 --bwa_bin $bwa_bin --samtools_bin $samtools_bin --maxThreads $maxThreads);
	print "Now executing: $command_extraction\n";
	system($command_extraction) and die "Command $command_extraction failed\n";
}
else
{
	my $host = hostname();
	# my $MHC_PRG_2_bin = (($host =~ /rescomp/) or ($host =~ /comp[ABC]/)) ? '../bin_cluster3/HLA-LA' : '../bin/HLA-LA';
	# my $MHC_PRG_2_bin = '../bin/HLA-LA';
	# my $MHC_PRG_2_bin = '/HLA-LA/bin/HLA-LA';
	my $MHC_PRG_2_bin = '/usr/local/bin/HLA-LA';


	my $previous_dir = getcwd;
	chdir($this_bin_dir) or die "Cannot cd into $this_bin_dir";

	die "Binary $MHC_PRG_2_bin not there!" unless(-e $MHC_PRG_2_bin);
	my $command_MHC_PRG = qq($MHC_PRG_2_bin --action HLA --maxThreads $maxThreads --sampleID $sampleID --outputDirectory $working_dir_thisSample --PRG_graph_dir $full_graph_dir --FASTQU $target_FASTQ_U_split --FASTQ1 $target_FASTQ_1 --FASTQ2 $target_FASTQ_2 --bwa_bin $bwa_bin --samtools_bin $samtools_bin --mapAgainstCompleteGenome $mapAgainstCompleteGenome --longReads $longReads);

	print "\nNow executing:\n$command_MHC_PRG\n";

	if(system($command_MHC_PRG) != 0)
	{
		die "HLA-LA execution not successful. Command was $command_MHC_PRG\n";
	}

	chdir($previous_dir) or die "Cannot cd into $previous_dir";
}

sub find_path
{
	my $id = shift;
	my $supplied_value = shift;
	my $forWhich = shift;

	if(defined $supplied_value)
	{
		die "Command-line supplied value/file for parameter $id not existing" unless(-e $supplied_value);
		return $supplied_value;
	}

	if(exists $paths_ini{$id})
	{
		foreach my $alternative (@{$paths_ini{$id}})
		{
			if(-e $alternative)
			{
				return $alternative;
			}
		}
	}

	if($forWhich)
	{
		my $which_output = `which $forWhich`;
		$which_output =~ s/[\n\r]//g;
		if($which_output and (-e $which_output))
		{
			return $which_output;
		}
	}

	die "I couldn't figure out a path for ${id}. Order of precedence: check for parameter --${id}; check paths.ini in $this_bin_dir for a key named $id; parse, if command string defined, the output of the command 'which ${forWhich}' ('which ' means that the command string is not defined).";
}
	#!/usr/bin/env perl

	# ./inferHLATypes.pl --BAM /gpfs1/well/gsk_hla/bam_output/AA02O9Q_Z2.bam --graph PRG_MHC_GRCh38_withIMGT --sampleID NA12878Direct

	use warnings;
	use strict;
	use FindBin;
	use File::Spec;
	use Getopt::Long;
	use Data::Dumper;
	use Sys::Hostname;
	use Cwd qw/getcwd abs_path/;

	$\| = 1;
	my $this_bin_dir = $FindBin::RealBin;

	my $_BAM;
	my $graph;
	my $GRAPHDIR;
	my $sampleID;
	my $qsub;
	my $samtools_bin;
	my $bwa_bin;
	my $java_bin;
	my $picard_sam2fastq_bin;
	my $workingDir_param;
	my $maxThreads = 1;
	my $moreReferencesDir;
	my $extractExonkMerCounts;
	my $longReads = 0;
	my $testing = 0;
	GetOptions (
	'BAM:s' => \$_BAM,
	'graph:s' => \$graph,
	'GRAPHDIR:s' => \$GRAPHDIR,
	'sampleID:s' => \$sampleID,
	'qsub:s' => \$qsub,
	'workingDir:s' => \$workingDir_param,
	'samtools_bin:s' => \$samtools_bin,
	'bwa_bin:s' => \$bwa_bin,
	'java_bin:s' => \$java_bin,
	'picard_sam2fastq_bin:s' => \$picard_sam2fastq_bin,
	'maxThreads:s' => \$maxThreads,
	'moreReferencesDir:s' => \$moreReferencesDir,
	'extractExonkMerCounts:s' => \$extractExonkMerCounts,
	'longReads:s' => \$longReads,
	'testing:s' => \$testing,
	);

	if ($extractExonkMerCounts)
	{
	warn "--extractExonkMerCounts 1: This feature is experimental and unlikely to work on your machine";
	die unless(-e '../exonkMerExtraction/GRCh38.forkMers');
	die unless(-e '../exonkMerExtraction/exonCoordinates_manual.txt.forExtraction');
	}

	unless((not $longReads) or ($longReads eq 'ont2d') or ($longReads eq 'pacbio'))
	{
	die "Please specify --longReads ont2d or --longReads pacbio";
	}

	my %paths_ini;
	my $paths_ini = $this_bin_dir . '/paths.ini';
	if(-e $paths_ini)
	{
	open(INI, '<', $paths_ini) or die "Cannot open $paths_ini";
	while(<INI>)
	{
	chomp;
	next unless($_);
	$_ =~ s/[\n\r]//g;
	next if($_ =~ /^\s+$/);
	die "Invalid line format in $paths_ini -- expect all lines to be either empty or key=value pairs" unless($_ =~ /^(.+)=(.*)$/);
	my $id = $1;
	my @alts = split(/,/, $2);
	$paths_ini{$id} = \@alts;
	}
	close(INI);
	}
	$samtools_bin = find_path('samtools_bin', $samtools_bin, 'samtools');
	$bwa_bin = find_path('bwa_bin', $bwa_bin, 'bwa');
	$java_bin = find_path('java_bin', $java_bin, 'java');
	$picard_sam2fastq_bin = find_path('picard_sam2fastq_bin', $picard_sam2fastq_bin, 'picard');

	my $FASTQ_extraction_command_part1;
	if($picard_sam2fastq_bin =~ /SamToFastq\.jar$/)
	{
	$FASTQ_extraction_command_part1 = qq($java_bin -Xmx10g -XX:-UseGCOverheadLimit -jar $picard_sam2fastq_bin);
	}
	elsif($picard_sam2fastq_bin =~ /picard-tools$/)
	{
	$FASTQ_extraction_command_part1 = qq($picard_sam2fastq_bin SamToFastq);
	}
	elsif($picard_sam2fastq_bin =~ /picard\.jar$/)
	{
	$FASTQ_extraction_command_part1 = qq($java_bin -Xmx10g -XX:-UseGCOverheadLimit -jar $picard_sam2fastq_bin SamToFastq);
	}
	elsif($picard_sam2fastq_bin =~ /picard$/)
	{
	$FASTQ_extraction_command_part1 = qq($picard_sam2fastq_bin SamToFastq);
	}
	else
	{
	die "I can't interpret the specified Picard command: $picard_sam2fastq_bin";
	}

	if($testing)
	{
	my $previous_dir = getcwd;
	chdir($this_bin_dir) or die "Cannot cd into $this_bin_dir";
	die "Binary ../bin/HLA-LA (from $this_bin_dir) not there!" unless(-e '../bin/HLA-LA');
	my $cmd_test = qq(../bin/HLA-LA --action testBinary);
	system($cmd_test) and die "HLA*LA test command $cmd_test failed";
	chdir($previous_dir) or die "Cannot chdir into $previous_dir";

	print "HLA-LA.pl test\n\n";
	print "\t", "samtools_bin", ": ", $samtools_bin, "\n";
	print "\t", "bwa_bin", ": ", $bwa_bin, "\n";
	print "\t", "java_bin", ": ", $java_bin, "\n";
	print "\t", "picard_sam2fastq_bin", ": ", $picard_sam2fastq_bin, "\n";
	exit 0;
	}


	my $working_dir;
	if($paths_ini{workingDir}[0] and not defined $workingDir_param)
	{
	$working_dir = $paths_ini{workingDir}[0];
	$working_dir =~ s/\$HLA\-LA\-DIR/$this_bin_dir/;
	}
	else
	{
	unless(defined $workingDir_param)
	{
	die "\n\nPlease specify a working directory via --workingDir.\n\nOutput for sample with ID \$sampleID will go a correspondingly named sub-directory of the working directory.\n\nFor example, if --workingDir is /path/working, and --sampleID is mySample, then the output will go into directory /path/working/mySample.\n\n";
	}
	$working_dir = $workingDir_param;
	}

	unless(-d $working_dir)
	{
	die "\n\nSpecified working directory $working_dir is either not present or not a directory.\n\nYou might have specified an invalid path via --workingDir.\n\n";
	}

	$working_dir = abs_path($working_dir);

	unless($sampleID =~ /^\w+$/)
	{
	die "Please use only alphanumeric characters - \\w+ - for --sampleID";
	}
	my $working_dir_thisSample = $working_dir . '/' . $sampleID;


	print "HLA-LA.pl\n\n";

	print "Identified paths:\n";
	print "\t", "samtools_bin", ": ", $samtools_bin, "\n";
	print "\t", "bwa_bin", ": ", $bwa_bin, "\n";
	print "\t", "java_bin", ": ", $java_bin, "\n";
	print "\t", "picard_sam2fastq_bin", ": ", $picard_sam2fastq_bin, "\n";
	print "\t", "General working directory", ": ", $working_dir, "\n";
	print "\t", "Sample-specific working directory", ": ", $working_dir_thisSample, "\n";

	print "\n";


	unless(-d $working_dir_thisSample)
	{
	mkdir($working_dir_thisSample) or die "Cannot mkdir $working_dir_thisSample";
	}

	my $samtools_version = `$samtools_bin --version` ;
	die "Can't parse samtools version output" unless($samtools_version =~ /samtools ([\d\.]+)/);
	$samtools_version = $1;
	my $samtools_version_numeric = $samtools_version;
	if($samtools_version_numeric =~ /^(\d+)\.(\d+)\.(\d+)$/)
	{
	$samtools_version_numeric = $1 . '.' . $2 . $3;
	}
	#unless($samtools_version_numeric >= 1.3)
	#{
	# die "I need samtools >=1.3";
	#}


	#my $BAM = File::Spec->abs2rel($_BAM);
	my $BAM = $_BAM;
	print "1343 BAM: $BAM \n";
	unless(-e $BAM)
	{
	die "BAM (or CRAM) $BAM (inferred from input $_BAM) not existing" unless(-e $BAM);
	unless((-e $BAM . '.bai') or (-e $BAM . '.crai'))
	{
	die "File $BAM does not appear to be indexed";
	}
	}

	my $full_graph_dir = $GRAPHDIR . '/' . $graph;
	my $known_references_dir = $full_graph_dir . '/knownReferences';
	unless(-e $full_graph_dir)
	{
	die "Graph directory $full_graph_dir not found - valid graph names are subdirectories of the graphs directory in the HLA-LA root";
	}
	unless((-e $full_graph_dir . '/sequences.txt') and ((-e $full_graph_dir . '/extendedReferenceGenomePath.txt') or (-e $full_graph_dir . '/extendedReferenceGenome/extendedReferenceGenome.fa')) and (-d $known_references_dir))
	{
	die "Graph directory $full_graph_dir does not seem to be complete - does this directory specify a valid graph for HLA-LA?";
	}

	# get index for this BAM
	my $command_idxstats = qq($samtools_bin idxstats $BAM);
	my $command_idxstats_output = `$command_idxstats`;
	unless($command_idxstats_output)
	{
	die "Didn't get a sensible output from command $command_idxstats";
	}
	my %BAM_idx;
	my @BAM_idx_contigOrder;
	my @idxstats_lines = split(/\n/, $command_idxstats_output);
	foreach my $l (@idxstats_lines)
	{
	my @l_fields = split(/\t/, $l);
	die unless(scalar(@l_fields) >= 1);
	die if(exists $BAM_idx{$l_fields[0]});
	$BAM_idx{$l_fields[0]} = $l_fields[1];
	push(@BAM_idx_contigOrder, $l_fields[0]);
	}

	my @files_references = glob($known_references_dir . '/*.txt');
	die "No known reference files in knownReferences ($full_graph_dir)?" unless(@files_references);
	my $additional_references_dir = $this_bin_dir . '/additionalReferences/' . $graph;
	if(-e $additional_references_dir)
	{
	my @additional_files_references = glob($additional_references_dir . '/*.txt');
	push(@files_references, @additional_files_references);
	}
	if($moreReferencesDir and (-e $moreReferencesDir))
	{
	my $mD = $moreReferencesDir . '/' . $graph;
	if(-d $mD)
	{
	my @even_more_files_references = glob($mD . '/*.txt');
	push(@files_references, @even_more_files_references);
	}
	}

	my @compatible_files;
	my %extractContigs_complete_byFile;
	my %extractContigs_partial_byFile;
	foreach my $f (@files_references)
	{
	open(F, '<', $f) or die "Cannot open $f";
	my $F_firstLine = <F>;
	chomp($F_firstLine);
	my @firstLine_fields = split(/\t/, $F_firstLine);
	my @expected_firstLine_fields = qw/contigID contigLength ExtractCompleteContig PartialExtraction_Start PartialExtraction_Stop/;
	die "Incorrect header for $f ($#firstLine_fields vs $#expected_firstLine_fields)" unless($#firstLine_fields == $#expected_firstLine_fields);
	for(my $i = 0; $i <= $#firstLine_fields; $i++)
	{
	die "Incorrect header for $f" unless($firstLine_fields[$i] eq $expected_firstLine_fields[$i]);
	}

	my $n_contigs = 0;
	my $n_contigs_matching = 0;
	while(<F>)
	{
	my $line = $_;
	chomp($line);
	next unless($line);
	my @line_fields = split(/\t/, $line, -1);
	$n_contigs++;
	if((exists $BAM_idx{$line_fields[0]}) and ($BAM_idx{$line_fields[0]} == $line_fields[1]))
	{
	$n_contigs_matching++;
	}
	else
	{
	# print "Mismatch $line_fields[0] - $line_fields[1] - " . $BAM_idx{$line_fields[0]} . "\n";
	}

	die if(($line_fields[2]) and (($line_fields[3]) and ($line_fields[4])));

	if($line_fields[2])
	{
	if($line_fields[0] eq '*')
	{
	$extractContigs_complete_byFile{$f}{$line_fields[0]} = 1;
	}
	else
	{
	$extractContigs_partial_byFile{$f}{$line_fields[0]} = [1, $line_fields[1]];
	}
	}

	if(($line_fields[3]) and ($line_fields[4]))
	{
	die if($line_fields[0] eq '*');
	die "Coordinate field $line_fields[3] has non-numeric characters" unless($line_fields[3] =~ /^\d+$/);
	die "Coordinate field $line_fields[4] has non-numeric characters" unless($line_fields[4] =~ /^\d+$/);

	$extractContigs_partial_byFile{$f}{$line_fields[0]} = [$line_fields[3], $line_fields[4]];
	}
	}
	close(F);

	if(($n_contigs_matching == $n_contigs) and ($n_contigs == scalar(@BAM_idx_contigOrder)))
	{
	push(@compatible_files, $f);
	}
	}

	if(scalar(@compatible_files) == 0)
	{
	die "Have found no compatible reference specifications in $known_references_dir - create a file for this BAM file and try again.";
	}
	if(scalar(@compatible_files) > 1)
	{
	die "Found more than one compatible reference file in $known_references_dir - a duplicate?\n\n".Dumper(\@compatible_files);
	}

	my $compatible_reference_file = $compatible_files[0];

	my @refIDs_for_extraction;
	foreach my $refID (@BAM_idx_contigOrder)
	{
	next if($refID eq '*');
	die if((exists $extractContigs_complete_byFile{$compatible_reference_file}{$refID}) and (exists $extractContigs_partial_byFile{$compatible_reference_file}{$refID}));
	die unless((not defined $extractContigs_complete_byFile{$compatible_reference_file}{$refID}) or ($extractContigs_complete_byFile{$compatible_reference_file}{$refID} eq '0') or ($extractContigs_complete_byFile{$compatible_reference_file}{$refID} eq '1'));
	if($extractContigs_complete_byFile{$compatible_reference_file}{$refID})
	{
	push(@refIDs_for_extraction, $refID);
	}
	if($extractContigs_partial_byFile{$compatible_reference_file}{$refID})
	{
	push(@refIDs_for_extraction, $refID . ':' . $extractContigs_partial_byFile{$compatible_reference_file}{$refID}[0] . '-' . $extractContigs_partial_byFile{$compatible_reference_file}{$refID}[1]);
	}
	}

	die "No contigs for extraction specified in $compatible_reference_file?" unless(scalar(@refIDs_for_extraction));

	my $target_extraction = $working_dir_thisSample . '/extraction.bam';


	my $target_extraction_mapped = $working_dir_thisSample . '/extraction_mapped.bam';
	my $extraction_command = qq($samtools_bin view -bo $target_extraction_mapped $BAM ).join(' ', @refIDs_for_extraction);
	print "Extract reads from ", scalar(@refIDs_for_extraction), " regions...\n";
	if(system($extraction_command) != 0)
	{
	die "Extraction command $extraction_command failed";
	}

	if($extractContigs_complete_byFile{$compatible_reference_file}{'*'})
	{
	my $target_extraction_unmapped = $working_dir_thisSample . '/extraction_unmapped.bam';

	my $extraction_command_unmapped = qq($samtools_bin view -b -f 4 $BAM > $target_extraction_unmapped);
	print "Extract unmapped reads...\n";
	print "extraction_command_unmapped=$extraction_command_unmapped\n";
	print "BAM: $BAM\n";

	if(system($extraction_command_unmapped) != 0)
	{
	die "Extraction command $extraction_command_unmapped failed";
	}

	unlink($target_extraction) if (-e $target_extraction);
	my $extraction_command_merge = qq($samtools_bin merge $target_extraction $target_extraction_mapped $target_extraction_unmapped);
	print "Merging...\n";
	if(system($extraction_command_merge) != 0)
	{
	die "Merge command $extraction_command_merge failed";
	}
	}
	else
	{
	my $mv_command = qq(mv $target_extraction_mapped $target_extraction);
	if(system($mv_command) != 0)
	{
	die "Move command $mv_command failed";
	}
	}

	my $index_command = qq($samtools_bin index $target_extraction);
	print "Indexing...\n";
	if(system($index_command) != 0)
	{
	die "Index command $index_command failed";
	}

	my $target_FASTQ_1 = $working_dir_thisSample . '/R_1.fastq';
	my $target_FASTQ_2 = $working_dir_thisSample . '/R_2.fastq';
	my $target_FASTQ_U = $working_dir_thisSample . '/R_U.fastq';
	my $target_FASTQ_U_split = $working_dir_thisSample . '/R_U.fastq.splitLongReads';
	my $FASTQ_extraction_command = qq($FASTQ_extraction_command_part1 VALIDATION_STRINGENCY=LENIENT I=$target_extraction F=$target_FASTQ_1 F2=$target_FASTQ_2 FU=$target_FASTQ_U 2>&1);

	print "Extract FASTQ...\n\t$FASTQ_extraction_command\n";
	my $FASTQ_extraction_output = `$FASTQ_extraction_command`;
	#if(($FASTQ_extraction_output =~ /Exception/) or ($FASTQ_extraction_output !~ /net.sf.picard.sam.SamToFastq done/))
	if(($FASTQ_extraction_output !~ /picard.sam.SamToFastq done/))
	{
	die "Picard output: \n\n" . $FASTQ_extraction_output . "\n\nExtraction command $FASTQ_extraction_command $! \n\nAbort because the Picard FASTQ extraction process might have failed. I think so because I could not find the string 'picard.sam.SamToFastq done' in the Picard output.\n\n";
	}

	if($longReads)
	{
	die "You activated --longReads, but the two files $target_FASTQ_1 and $target_FASTQ_2 (which store paired-end reads) are not empty - this is weird, and I will abort." unless(((-s $target_FASTQ_1) == 0) && ((-s $target_FASTQ_2) == 0));
	unless((-s $target_FASTQ_U) > 0)
	{
	die "You activated --longReads, but my attempt to extract long unpaired reads from the specified input BAM failed. Abort.";
	}

	open(INLONG, '<', $target_FASTQ_U) or die;
	open(OUTLONG, '>', $target_FASTQ_U_split) or die;
	while(<INLONG>)
	{
	chomp;
	my $readID = $_;
	die unless(substr($readID, 0, 1) eq '@');
	my $sequence = <INLONG>; chomp($sequence);
	my $plus = <INLONG>;
	my $qualities = <INLONG>; chomp($qualities);
	die unless(substr($plus, 0, 1) eq '+');
	die unless(length($sequence) == length($qualities));


	if(length($sequence) < 50000)
	{
	print OUTLONG $readID, "\n", $sequence, "\n", "+\n", $qualities, "\n";
	}
	else
	{
	my $runningI = 0;
	while(length($sequence))
	{
	die unless(length($sequence) == length($qualities));

	my $thisPart_readID = '>rP' . $runningI . substr($readID, 1);

	my $extractionLength = (length($sequence) > 50000) ? 50000 : length($sequence);
	print OUTLONG $thisPart_readID, "\n", substr($sequence, 0, $extractionLength), "\n", "+\n", substr($qualities, 0, $extractionLength), "\n";

	substr($sequence, 0, $extractionLength) = '';
	substr($qualities, 0, $extractionLength) = '';

	$runningI++;
	}
	}
	}
	close(INLONG);
	close(OUTLONG);


	}
	else
	{
	die "You didn't activate --longReads, but the two files $target_FASTQ_1 and $target_FASTQ_2 (which store paired-end reads) are empty - this is weird, and I will abort." unless(((-s $target_FASTQ_1) > 0) && ((-s $target_FASTQ_2) > 0));
	}

	#if(system($FASTQ_extraction_command) != 0)
	#{
	#}

	my $mapAgainstCompleteGenome = ($extractContigs_complete_byFile{$compatible_reference_file}{'*'}) ? 1 : 0;
	#$mapAgainstCompleteGenome = 0;

	if($extractExonkMerCounts)
	{
	die if($longReads);
	die unless(-e '../exonkMerExtraction/GRCh38.forkMers');
	die unless(-e '../exonkMerExtraction/exonCoordinates_manual.txt');

	my $command_extraction = qq(perl extractkMerCounts.pl --sampleID $sampleID --outputDirectory $working_dir_thisSample --referenceGenome ../exonkMerExtraction/GRCh38.forkMers --exonCoordinates ../exonkMerExtraction/exonCoordinates_manual.txt --FASTQ1 $target_FASTQ_1 --FASTQ2 $target_FASTQ_2 --bwa_bin $bwa_bin --samtools_bin $samtools_bin --maxThreads $maxThreads);
	print "Now executing: $command_extraction\n";
	system($command_extraction) and die "Command $command_extraction failed\n";
	}
	else
	{
	my $host = hostname();
	# my $MHC_PRG_2_bin = (($host =~ /rescomp/) or ($host =~ /comp[ABC]/)) ? '../bin_cluster3/HLA-LA' : '../bin/HLA-LA';
	# my $MHC_PRG_2_bin = '../bin/HLA-LA';
	# my $MHC_PRG_2_bin = '/HLA-LA/bin/HLA-LA';
	my $MHC_PRG_2_bin = '/usr/local/bin/HLA-LA';


	my $previous_dir = getcwd;
	chdir($this_bin_dir) or die "Cannot cd into $this_bin_dir";

	die "Binary $MHC_PRG_2_bin not there!" unless(-e $MHC_PRG_2_bin);
	my $command_MHC_PRG = qq($MHC_PRG_2_bin --action HLA --maxThreads $maxThreads --sampleID $sampleID --outputDirectory $working_dir_thisSample --PRG_graph_dir $full_graph_dir --FASTQU $target_FASTQ_U_split --FASTQ1 $target_FASTQ_1 --FASTQ2 $target_FASTQ_2 --bwa_bin $bwa_bin --samtools_bin $samtools_bin --mapAgainstCompleteGenome $mapAgainstCompleteGenome --longReads $longReads);

	print "\nNow executing:\n$command_MHC_PRG\n";

	if(system($command_MHC_PRG) != 0)
	{
	die "HLA-LA execution not successful. Command was $command_MHC_PRG\n";
	}

	chdir($previous_dir) or die "Cannot cd into $previous_dir";
	}

	sub find_path
	{
	my $id = shift;
	my $supplied_value = shift;
	my $forWhich = shift;

	if(defined $supplied_value)
	{
	die "Command-line supplied value/file for parameter $id not existing" unless(-e $supplied_value);
	return $supplied_value;
	}

	if(exists $paths_ini{$id})
	{
	foreach my $alternative (@{$paths_ini{$id}})
	{
	if(-e $alternative)
	{
	return $alternative;
	}
	}
	}

	if($forWhich)
	{
	my $which_output = `which $forWhich`;
	$which_output =~ s/[\n\r]//g;
	if($which_output and (-e $which_output))
	{
	return $which_output;
	}
	}

	die "I couldn't figure out a path for ${id}. Order of precedence: check for parameter --${id}; check paths.ini in $this_bin_dir for a key named $id; parse, if command string defined, the output of the command 'which ${forWhich}' ('which ' means that the command string is not defined).";
	}