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HLA-ASM.pl
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#!/usr/bin/env perl
BEGIN {
use FindBin;
push(@INC, $FindBin::Bin);
push(@INC, $FindBin::RealBin);
}
use warnings;
use strict;
use FindBin;
use Data::Dumper;
use Storable qw/store retrieve dclone/;
use List::MoreUtils qw/mesh all/;
use List::Util qw/max min/;
eval "use Text::LevenshteinXS qw(distance);";
if($@)
{
warn "Text::LevenshteinXS could not be loaded, falling back onto Text::Levenshtein -- install Text::LevenshteinXS for higher performance.";
eval "use Text::Levenshtein qw(distance);";
if($@)
{
die "Text::Levenshtein could also not be loaded";
}
}
use Getopt::Long;
use Cwd qw/getcwd abs_path/;
use findPath;
my $scriptPath = $FindBin::Bin;
my $includes=" -I" . join(" -I", @INC);
$| = 1;
my $this_bin_dir = $FindBin::RealBin;
# Test command
# perl HLA-ASM.pl --assembly_fasta /data/projects/phillippy/scratch/trioCanu/NA12878/mhc/nanopolish/dad_methylation/dad_nm_mhc.fasta --sampleID NA12878_nanopolish_dad_methylation --truth reference_HLA_ASM/truth_NA12878.txt
my $sampleID;
my $assembly_fasta;
my $tolerantMissing = 0;
my $truthFile;
my $samtools_bin;
my $bwa_bin;
my $nucmer_bin;
my $dnadiff_bin;
my $workingDir_param;
my $testing;
my $use_minimap2 = 0;
my $max_report_edit_distance = 150;
GetOptions (
'assembly_fasta:s' => \$assembly_fasta,
'sampleID:s' => \$sampleID,
'tolerantMissing:s' => \$tolerantMissing,
'truthFile:s' => \$truthFile,
'samtools_bin:s' => \$samtools_bin,
'bwa_bin:s' => \$bwa_bin,
'nucmer_bin:s' => \$nucmer_bin,
'dnadiff_bin:s' => \$dnadiff_bin,
'workingDir:s' => \$workingDir_param,
'testing:s' => \$testing,
'use_minimap2:s' => \$use_minimap2,
'max_report_edit_distance:s' => \$max_report_edit_distance,
);
$samtools_bin = findPath::find_path('samtools_bin', $samtools_bin, 'samtools');
$bwa_bin = findPath::find_path('bwa_bin', $bwa_bin, 'bwa');
$nucmer_bin = findPath::find_path('nucmer_bin', $nucmer_bin, 'nucmer');
$dnadiff_bin = findPath::find_path('dnadiff_bin', $dnadiff_bin, 'dnadiff');
findPath::check_samtools($samtools_bin);
if($testing)
{
print "HLA*ASM self-test\n";
print "Identified paths: $samtools_bin $bwa_bin $nucmer_bin $dnadiff_bin\n";
exit(0);
}
unless($assembly_fasta and $sampleID)
{
die "Please specify parameters --assembly_fasta and --sampleID. --assembly_fasta should specify a path to a unified FASTA of your assembly with unique contig IDs, and sampleID should be an alphanumeric sample ID";
}
die "File --assembly_fasta not existing" unless(-e $assembly_fasta);
die "Please specify an alphanumeric --sampleID" unless($sampleID =~ /^\w+$/);
if($truthFile)
{
die "--truthFile $truthFile not existing" unless(-e $truthFile);
$truthFile = abs_path($truthFile);
}
$assembly_fasta = abs_path($assembly_fasta);
chdir($this_bin_dir) or die "Cannot chdir into $this_bin_dir";
my $GRCh38_primary = 'reference_HLA_ASM/hg38.primary.fna';
die "Reference reference_HLA_ASM/hg38.primary.fna not existing -- have you downloaded and installed the HLA-ASM data package?" unless(-e $GRCh38_primary);
my %classI = map {$_ => 1} qw/HLA-A HLA-B HLA-C/;
my %classII = map {$_ => 1} qw/HLA-DQA1 HLA-DQB1 HLA-DRB1 HLA-DRB3 HLA-DRB4/;
my $working_dir = findPath::get_working_dir($workingDir_param);
unless(-e $working_dir)
{
mkdir($working_dir) or die "Cannot mkdir $working_dir";
}
my $temp_alignments_dir = $working_dir . '/' . $sampleID . '/temp_alignments_separate/';
unless(-e $working_dir . '/' . $sampleID)
{
mkdir( $working_dir . '/' . $sampleID) or die "Cannot mkdir " . $working_dir . '/' . $sampleID;
}
unless(-e $temp_alignments_dir)
{
mkdir($temp_alignments_dir) or die "Cannot mkdir $temp_alignments_dir";
}
mkdir('temp') unless(-d 'temp');
die unless(-d 'temp');
my $fn_assembly_path = $temp_alignments_dir . '/assemblyPath.txt';
if(not -e $fn_assembly_path)
{
open(ASSEMBLYPATH, '>', $fn_assembly_path) or die "Cannot open $fn_assembly_path";
print ASSEMBLYPATH $assembly_fasta, "\n";
close(ASSEMBLYPATH);
}
else
{
open(ASSEMBLYPATH, '<', $fn_assembly_path) or die "Cannot open $fn_assembly_path";
my $firstLine = <ASSEMBLYPATH>;
chomp($firstLine);
close(ASSEMBLYPATH);
unless($firstLine eq $assembly_fasta)
{
die "The file $fn_assembly_path indicates that you already used this sample ID, but with a different assembly file. As I am not smart enough to figure out which files need to be re-generated under these circumstances, it's best to delete the whole directory $temp_alignments_dir and start over. Alternatively, just use a different sample ID.";
}
}
my $verbose_reading = 0;
my %relevantRegions = (
'MHC' => ['CM000668.2', 28510120, 33480577, 'reference_HLA_ASM/MHC8.mfa'],
# 'KIR' => ['CM000681.2', 54025634, 55084318, 'KIR36_plus.mfa'],
);
my %G_full_alleles;
my %G_mapper_unambigious;
my %G_mapper_multiples;
my %G_to_underlyingAlleles;
my %alleles_to_fullGAmbiguity;
readG();
my %sample_truth;
if($truthFile)
{
open(TRUTH, '<', $truthFile) or die "Cannot open $truthFile";
while(<TRUTH>)
{
my $line = $_;
chomp($line);
$line =~ s/[\n\r]//g;
next unless($line);
my @line_fields = split(/\t/, $line);
die "Invalid line in $truthFile - expect tab-separated: HLA-LOCUS allele1 allele2. Example: HLA-A\t01:01:01:01\t11:01:01:01" unless(scalar(@line_fields) == 3);
die "Invalid line in $truthFile - expect tab-separated: HLA-LOCUS allele1 allele2. Example: HLA-A\t01:01:01:01\t11:01:01:01" unless($line_fields[0] =~ /^HLA-/);
die "Duplicate locus in $truthFile for $line_fields[0]?" if(exists $sample_truth{$line_fields[0]});
my @alleles_tr;
foreach my $allele ($line_fields[1], $line_fields[2])
{
die "Invalid line in $truthFile - expect tab-separated: HLA-LOCUS allele1 allele2. Example: HLA-A\t01:01:01G\t11:01:01:01.\n\nTyping is ALWAYS carried out a G group resolution." unless($line_fields[1] =~ /\:/);
my $locus_for_gGroupLookup = $line_fields[0];
$locus_for_gGroupLookup =~ s/HLA-/HLA/;
die "Unknown locus $line_fields[0]" unless(exists $G_to_underlyingAlleles{$locus_for_gGroupLookup});
die "Can't resolve allele $line_fields[0] $allele at G-group resolution" unless(defined $G_to_underlyingAlleles{$locus_for_gGroupLookup}{$allele});
my $allele_tr = $G_to_underlyingAlleles{$locus_for_gGroupLookup}{$allele}[0];
$allele_tr =~ s/^\S+\*//;
push(@alleles_tr, $allele_tr);
}
$sample_truth{$line_fields[0]} = \@alleles_tr;
}
close(TRUTH);
}
my $fn_contigs = $assembly_fasta;
my $contigs_href = readFASTA($fn_contigs, 1);
validate_assembly($assembly_fasta);
my $mcoords = produce_nucmer($assembly_fasta);
my $fn_nucmer_wcoords = $mcoords;
open(DOTPLOTDATA, '>', $temp_alignments_dir . '/dotPlot.tab') or die;
open(EDITDISTANCE, '>', $temp_alignments_dir . '/editDistances.tab') or die;
open(CONTIGLENGTHS, '>', $temp_alignments_dir . '/refContigLengths.tab') or die;
open(GENEPOSITIONS, '>', $temp_alignments_dir . '/genePositions.tab') or die;
open(GENESEQUENCES, '>', $temp_alignments_dir . '/geneSequences.txt') or die;
print DOTPLOTDATA join("\t", "region", "contigID", "refID", "refCoordinate", "contigCoordinate"), "\n";
print EDITDISTANCE join("\t", "region", "contigID", "refID", "editDistance"), "\n";
print CONTIGLENGTHS join("\t", "region", "refID", "length"), "\n";
print GENEPOSITIONS join("\t", "contigID", "contigStrand", "gene", "definedFromRefID", "startInContig_0Based_relativeToSpecifiedStrand", "stopInContig_0Based_relativeToSpecifiedStrand", "capturedParts"), "\n";
my %overlappingContig_strand_called;
my %results;
foreach my $region (sort keys %relevantRegions)
{
my $regionInfo = $relevantRegions{$region};
my $references_href = readFASTA($regionInfo->[3]);
my $exon_allele_alignments_href;
#my $genomic_allele_alignments_withPARTS_href;
my %sequenceExtractionCoordinates_start;
my %sequenceExtractionCoordinates_stop;
my %sequenceExtractionCoordinates_refDBStrand;
my %alleles_2_features;
if($region eq 'MHC')
{
my $exon_allele_alignments_href_file = 'temp/_exon_allele_alignments_href';
#my $genomic_allele_alignments_withPARTS_href_file = 'temp/_genomic_allele_alignments_withPARTS_href';
if(-e $exon_allele_alignments_href_file)
{
$exon_allele_alignments_href = retrieve $exon_allele_alignments_href_file;
#$genomic_allele_alignments_withPARTS_href = retrieve $genomic_allele_alignments_withPARTS_href_file;
}
else
{
$exon_allele_alignments_href = read_exon_alignments(1);
store($exon_allele_alignments_href, $exon_allele_alignments_href_file);
#$genomic_allele_alignments_withPARTS_href = read_genomic_alignments(1);
#store($genomic_allele_alignments_withPARTS_href, $genomic_allele_alignments_withPARTS_href_file);
}
open(EXONS, '<', $regionInfo->[3] . '.exons') or die;
my $exons_headerLine = <EXONS>;
chomp($exons_headerLine);
my @exons_header_fields = split(/\t/, $exons_headerLine);
while(<EXONS>)
{
my $line = $_;
chomp($line);
next unless($line);
my @line_fields = split(/\t/, $line);
die unless($#line_fields == $#exons_header_fields);
my %line = (mesh @exons_header_fields, @line_fields);
push(@{$sequenceExtractionCoordinates_start{$line{refID}}{$line{Start_0based}}}, [$line{geneName}, $line{subComponent}]);
push(@{$sequenceExtractionCoordinates_stop{$line{refID}}{$line{Stop_0based}}}, [$line{geneName}, $line{subComponent}]);
if(not defined $sequenceExtractionCoordinates_refDBStrand{$line{refID}}{$line{geneName}})
{
$sequenceExtractionCoordinates_refDBStrand{$line{refID}}{$line{geneName}} = $line{alleleDatabase_strand};
}
die unless($sequenceExtractionCoordinates_refDBStrand{$line{refID}}{$line{geneName}} eq $line{alleleDatabase_strand});
}
close(EXONS);
foreach my $locus (keys %$exon_allele_alignments_href)
{
foreach my $allele (keys %{$exon_allele_alignments_href->{$locus}})
{
my @exons = split(/\|/, $exon_allele_alignments_href->{$locus}{$allele});
die unless(scalar(@exons) > 1);
for(my $exonI = 1; $exonI <= scalar(@exons); $exonI++)
{
my $exonSeq = $exons[$exonI-1];
$exonSeq =~ s/[\-_]//g;
$exonSeq = uc($exonSeq);
die Dumper("Weird exon sequence", $exonSeq) unless($exonSeq =~ /^[ACGTN]*$/);
$alleles_2_features{$locus}{$allele}{'exon'.$exonI} = $exonSeq;
}
}
}
foreach my $DRBallele (keys %{$alleles_2_features{'HLA-DRB'}})
{
die unless($DRBallele =~ /(DRB\d)/);
my $realLocus = $1;
$alleles_2_features{'HLA-'.$realLocus}{$DRBallele} = $alleles_2_features{'HLA-DRB'}{$DRBallele};
}
delete $alleles_2_features{'HLA-DRB'};
foreach my $locus (keys %sample_truth)
{
foreach my $truthAllele (@{$sample_truth{$locus}})
{
my $truth_key = $locus . '*' . $truthAllele;
$truth_key =~ s/HLA\-//;
die Dumper("Can't find sequence for $locus $truth_key (specified in file $truthFile)", ["Examples", (keys %{$alleles_2_features{$locus}})[0 .. 9]], [keys %alleles_2_features]) unless(exists $alleles_2_features{$locus}{$truth_key});
}
}
#open(DRB3, '>', 'NA12878_DRB3_sequence.fa') or die;
#my $DRB3_sequence = $exon_allele_alignments_href->{'HLA-DRB'}{'DRB3*01:01:02:01'};
#$DRB3_sequence =~ s/-//g;
#$DRB3_sequence =~ s/_//g;
#$DRB3_sequence =~ s/\|//g;
#print DRB3 '>DRB3*01:01:02:01', "\n";
#print DRB3 $DRB3_sequence, "\n";
#close(DRB3);
#exit;
# die Dumper(keys %{$alleles_2_features{'HLA-DRB3'}});
# $alleles_2_features{'HLA-DRB1'} = $alleles_2_features{'HLA-DRB'};
}
my %overlappingContigs;
my %overlappingContigs_idty;
my %overlappingContig_strands;
my %strand_per_originalContig;
my %strand_per_haplotig;
my %alphanumeric_keys;
foreach my $refContig (keys %$references_href)
{
my $refID_alphanumeric = $refContig;
$refID_alphanumeric =~ s/\W//g;
die Dumper("Duplicate ID?", $refID_alphanumeric, \%alphanumeric_keys) if($alphanumeric_keys{$refID_alphanumeric});
$alphanumeric_keys{$refID_alphanumeric}++;
}
my %alignments_per_refContig;
foreach my $refContigID (sort keys %$references_href)
{
next if($refContigID eq 'gi|568335997|gb|KI270758.1| Homo sapiens chromosome 6 genomic contig, GRCh38 reference assembly alternate locus group ALTREFLOCI8');
my $refContigSequence = $references_href->{$refContigID};
print CONTIGLENGTHS join("\t", $region, $refContigID, length($refContigSequence)), "\n";
}
if($fn_nucmer_wcoords)
{
open(COORDS, '<', $fn_nucmer_wcoords) or die "Cannot open $fn_nucmer_wcoords";
while(<COORDS>)
{
my $line = $_;
chomp($line);
next unless($line);
my @line_fields = split(/\t/, $line);
my $refID = $line_fields[11];
my $contigID = $line_fields[12];
my $refStart = $line_fields[0];
my $refStop = $line_fields[1];
my $contigStart = $line_fields[2];
my $contigStop = $line_fields[3];
my $strand = ($contigStart < $contigStop) ? '+' : '-';
my $identity = $line_fields[6];
next unless($refID eq $regionInfo->[0]);
if(overlap($regionInfo->[1], $regionInfo->[2], $refStart, $refStop))
{
my $useKey = $contigID;
if(exists $contigs_href->{$useKey . '_pilon'})
{
$useKey = $useKey . '_pilon';
}
elsif($contigs_href->{$useKey . '_pilon_pilon'})
{
$useKey = $useKey . '_pilon_pilon';
}
$overlappingContigs{$useKey}++;
$overlappingContigs_idty{$useKey} = $identity;
$overlappingContig_strands{$useKey}{$strand}++;
}
}
close(COORDS);
print "${region}-overlapping contigs:\n";
print join("\n", map {' - ' . $_ . ' [' . $overlappingContigs_idty{$_} . '] - ' . (exists $overlappingContig_strands{$_}{'+'} ? $overlappingContig_strands{$_}{'+'} : 0) . '+ / ' . (exists $overlappingContig_strands{$_}{'-'} ? $overlappingContig_strands{$_}{'-'} : 0) . '-'} keys %overlappingContigs), "\n";
foreach my $contigID (keys %overlappingContigs)
{
my @strands = keys(%{$overlappingContig_strands{$contigID}});
@strands = reverse sort {$overlappingContig_strands{$contigID}{$a} <=> $overlappingContig_strands{$contigID}{$b}} @strands;
my $strand = $strands[0];
unless(scalar(@strands) == 1)
{
warn join("\t", $contigID, @strands), "\n";
warn "No enquivocal strand call for $contigID - now $strand - , check manually!";
}
$strand_per_originalContig{$contigID} = $strand;
}
# foreach my $contigID (keys %overlappingContigs)
# {
# foreach my $haplotigID (keys %haplotigs)
# {
# if(substr($haplotigID, 0, length($contigID)) eq $contigID)
# {
# $overlappingContigs{$haplotigID} = 1;
# my $haplotigSequence = $haplotigs{$haplotigID};
# my $jaccard_plus = kMerSharingSubset($contigs_href->{$contigID}, $haplotigSequence);
# my $jaccard_minus = kMerSharingSubset($contigs_href->{$contigID}, reverseComplement($haplotigSequence));
# print "Contig $contigID [", length($contigs_href->{$contigID}), "], haplotig $haplotigID [", length($haplotigSequence), "], plus $jaccard_plus, minus $jaccard_minus \n";
# my $haplotig_strand_relative_to_originalContig = ($jaccard_plus > $jaccard_minus) ? '+' : '-';
# my $haplotig_strand_absolute;
# if($haplotig_strand_relative_to_originalContig eq '+')
# {
# $haplotig_strand_absolute = $strand_per_originalContig{$contigID};
# }
# else
# {
# $haplotig_strand_absolute = ($strand_per_originalContig{$contigID} eq '-') ? '+' : '-';
# }
# $strand_per_haplotig{$haplotigID} = $haplotig_strand_absolute;
# $contigs_href->{$haplotigID} = $haplotigSequence;
# }
# }
# }
}
else
{
%overlappingContigs = map {$_ => 1} keys %$contigs_href;
%strand_per_originalContig = map {$_ => '+'} keys %$contigs_href;
}
# todo get rid of!
# $strand_per_originalContig{'1_tig00000010_B_pilon'} = '-';
# $strand_per_originalContig{'0_tig00000049_A_pilon'} = '-';
# $strand_per_originalContig{'1_tig00000003_B_pilon'} = '-';
# $strand_per_originalContig{'0_tig00000001_A_pilon'} = '-';
# $strand_per_originalContig{'0_tig00000022_A_pilon'} = '-';
# $strand_per_originalContig{'1_tig00000012_B_pilon'} = '-';
# $strand_per_originalContig{'0_tig00000008_A_pilon'} = '-';
# die Dumper((sort keys %overlappingContigs), \%strand_per_originalContig);
foreach my $contigID (sort keys %overlappingContigs)
{
# next unless($contigID eq 'tig00019339_pilon');
# next unless($contigID eq 'tig00007299_pilon');
unless(exists $contigs_href->{$contigID})
{
if($tolerantMissing)
{
warn "Contig $contigID undefined?";
next;
}
else
{
die "Contig $contigID undefined?";
}
}
my $contigID_alphanumeric = $contigID;
$contigID_alphanumeric =~ s/\W//g;
die if($alphanumeric_keys{$contigID});
$alphanumeric_keys{$contigID}++;
my $strand;
# if(not exists $haplotigs{$contigID})
{
$strand = $strand_per_originalContig{$contigID};
}
# else
# {
# $strand = $strand_per_haplotig{$contigID};
# }
die unless(defined $strand);
$overlappingContig_strand_called{$region}{$contigID} = $strand;
my $contigSequence = $contigs_href->{$contigID};
if(defined $strand and ($strand eq '-'))
{
$contigSequence = reverseComplement($contigSequence);
}
my %alignments_per_refContig;
foreach my $refContigID (sort keys %$references_href)
{
next if($refContigID eq 'gi|568335997|gb|KI270758.1| Homo sapiens chromosome 6 genomic contig, GRCh38 reference assembly alternate locus group ALTREFLOCI8');
my $refContigSequence = $references_href->{$refContigID};
my $refID_alphanumeric = $refContigID;
$refID_alphanumeric =~ s/\W//g;
my $fn_output = $temp_alignments_dir . '/' . $refID_alphanumeric . '_' . $contigID_alphanumeric;
my $T = '';
my $Q = '';
my $TStart_0based;
my $TStop_0based;
my $QStart_0based;
my $QStop_0based;
my $score;
my $alignmentStrand;
my $aligner = 'bwa';
if($aligner eq 'edlib')
{
unless(-e $fn_output)
{
writeFASTA('_contig', {contig => $contigSequence});
writeFASTA('_ref', {ref => $refContigSequence});
print "Aligning $contigID [${strand}]vs $refContigID \n";
my $edlib_cmd = qq(/data/projects/phillippy/software/edlib/build/bin/aligner -n 1 -m HW -p _contig _ref > $fn_output);
system($edlib_cmd);
}
{
open(F, '<', $fn_output) or die "Cannot open $fn_output";
while(<F>)
{
my $line = $_;
if($line =~ /score = (\d+)/)
{
die "More than one alignment?" if(defined $score);
$score = $1;
}
else
{
if($line =~ /^(T|Q)\: (\w+) \((\d+) - (\d+)\)/)
{
my $TQ = $1;
my $alignment = $2;
my $start = $3;
my $stop = $4;
if($TQ eq 'T')
{
$T .= $alignment;
$TStart_0based = $start if(not defined $TStart_0based);
$TStop_0based = $stop;
}
else
{
die unless($TQ eq 'Q');
$Q .= $alignment;
$QStart_0based = $start if(not defined $QStart_0based);
$QStop_0based = $stop;
}
}
}
}
close(F);
}
}
elsif($aligner eq 'nucmer')
{
die "Not fully implemented";
my $alignment_f = $fn_output . '.alignment';
print "Alignment file $alignment_f\n";
unless(-e $alignment_f)
{
print "Re-computing...\n";
writeFASTA('_contig', {contig => $contigSequence});
writeFASTA('_ref', {ref => $refContigSequence});
print "Aligning $contigID [${strand}]vs $refContigID \n";
my $mummer_cmd = qq(/data/projects/phillippy/software/MUMmerSensitive/nucmer -p $fn_output _ref _contig);
system($mummer_cmd) and die "Nucmer command $mummer_cmd failed";
my $delta_file = $fn_output . '.delta';
my $delta_file_g = $fn_output . '.delta.g';
my $filter_command = "delta-filter -g $delta_file > $delta_file_g";
system($filter_command) and die "Nucmer command $mummer_cmd failed";
my $alignment_command = "show-aligns -r $delta_file_g ref contig > $alignment_f";
system($alignment_command) and die "Nucmer command $alignment_command failed";
}
open(ALIGNMENT, '<', $alignment_f) or die "Cannot open $alignment_f";
while(<ALIGNMENT>)
{
my $line = $_;
chomp($line);
next unless($line);
my $inAlignment = 0;
if($line =~ /BEGIN alignment \[ ((\+1)|(-1)) (\d+) - (\d+) \| ((\+1)|(-1)) (\d+) - (\d+) \]/)
{
my $plusMinus_1 = $1;
my $start_ref = $4;
my $stop_ref = $5;
my $plusMinus_2 = $6;
my $start_seq = $9;
my $stop_seq = $10;
# warn Dumper($plusMinus_1, $start_ref, $stop_ref, $plusMinus_2, $start_seq, $stop_seq, $line);
die unless($plusMinus_1 eq '+1');
die unless($plusMinus_2 eq '+1');
die unless($start_ref < $stop_ref);
die unless($start_seq < $stop_seq);
$inAlignment = 1;
if(not defined $TStart_0based)
{
$TStart_0based = $start_ref - 1;
$QStart_0based = 0;
if($start_seq != 1)
{
my $missing_gaps = $start_seq - 1;
$T .= ('-' x $missing_gaps);
die unless(length($T) == $missing_gaps);
$Q .= substr($contigSequence, 0, $missing_gaps);
}
}
$TStop_0based = $stop_ref - 1;
if(defined $QStop_0based)
{
my $noGap_query = $Q;
$noGap_query =~ s/-//g;
die unless(length($noGap_query) == ($QStop_0based+1));
die Dumper($start_seq, $QStop_0based) unless(($start_seq-1) > $QStop_0based);
my $missing_query_gaps_in_between = ($start_seq-1) - $QStop_0based - 1;
$Q .= substr($contigSequence, $QStop_0based+1, $missing_query_gaps_in_between);
$T .= ('-' x $missing_query_gaps_in_between);
my $missing_ref_sequence = ($start_ref-1) - $TStop_0based - 1;
$T .= substr($refContigSequence, $TStop_0based + 1, $missing_ref_sequence);
$Q .= ('-' x $missing_ref_sequence);
}
$QStop_0based = $stop_seq - 1;
}
elsif($line =~ /END alignment/)
{
$inAlignment = 0;
}
else
{
if($line =~ /^\d+\s+([\w\.]+)/)
{
my $ref_seq = $1;
die "Weird sequence: '$ref_seq'" unless($ref_seq =~ /^[ACGTN\.]+$/i);
$ref_seq =~ s/\./-/g;
my $nextLine = <ALIGNMENT>;
chomp($nextLine);
die "Weird nextLine $nextLine" unless($nextLine =~ /^\d+\s+([\w\.]+)/);
my $query_seq = $1;
die unless($query_seq =~ /^[ACGTN\.]+$/i);
$query_seq =~ s/\./-/g;
$Q .= $query_seq;
$T .= $ref_seq;
}
}
}
if($QStop_0based != (length($contigSequence)-1))
{
my $missing_gaps_end = (length($contigSequence) - 1) - $QStop_0based;
die unless($missing_gaps_end > 0);
$Q .= substr($contigSequence, $QStop_0based+1);
$T .= ('-' x $missing_gaps_end);
}
my $Q_noGap = $Q;
my $T_noGap = $T;
$Q_noGap =~ s/-//g;
$T_noGap =~ s/-//g;
die unless(uc($Q_noGap) eq uc($contigSequence));
die unless(length($T_noGap) == ($TStop_0based - $TStart_0based + 1));
die unless(uc($T_noGap) eq uc(substr($refContigSequence, $TStart_0based, $TStop_0based - $TStart_0based + 1)));
}
elsif($aligner eq 'bwa')
{
my $fn_output_refined = $fn_output . '.2';
# if(($refContigID =~ /pgf/i) and ($contigID_alphanumeric =~ /000000F/i))
# {
# writeFASTA($temp_alignments_dir . '/_ref', {ref => $refContigSequence});
# writeFASTA($temp_alignments_dir . '/_contig', {contig => $contigSequence});
# my $cmd_align = qq(perl $includes $scriptPath/globalAlignment.pl --reference ${temp_alignments_dir}/_ref --query ${temp_alignments_dir}/_contig --output $fn_output --samtools_bin $samtools_bin --bwa_bin $bwa_bin);
# die Dumper($refContigID, $contigID_alphanumeric, $cmd_align);
# }
unless(-e $fn_output)
{
my $fn_ref = $temp_alignments_dir . '/' . $refID_alphanumeric . '_' . $contigID_alphanumeric . '_ref';
my $fn_contig = $temp_alignments_dir . '/' . $refID_alphanumeric . '_' . $contigID_alphanumeric . '_contig';
writeFASTA($fn_ref, {ref => $refContigSequence});
writeFASTA($fn_contig, {contig => $contigSequence});
my $cmd_align = qq(perl $includes $scriptPath/globalAlignment.pl --use_minimap2 $use_minimap2 --reference $fn_ref --query $fn_contig --output $fn_output --samtools_bin $samtools_bin --bwa_bin $bwa_bin);
print "Command: $cmd_align\n";
system($cmd_align) and die "Command $cmd_align failed";
}
unless(1 or (-e $fn_output_refined))
{
open(ALIGNMENT, '<', $fn_output) or die "Cannot open $fn_output";
my $l1 = <ALIGNMENT>; chomp($l1);
my $l2 = <ALIGNMENT>; chomp($l2);
my $l3 = <ALIGNMENT>; chomp($l3);
close(ALIGNMENT);
my $fn_forMAFFT = $fn_output_refined . '.forMAFFT';
my $fn_fromMAFFT = $fn_output_refined . '.fromMAFFT';
my $alignmentLength_before = length($l2);
$l2 =~ s/-//g;
$l3 =~ s/-//g;
writeFASTA($fn_forMAFFT, {'ref'=> $l2, 'contig' => $l3});
my $mafft_bin = '/data/projects/phillippy/projects/rDNA/mafft/mafft-7.273-with-extensions/install/bin/mafft';
my $cmd_mafft = qq($mafft_bin --retree 2 --maxiterate 2 $fn_forMAFFT > $fn_fromMAFFT);
if(system($cmd_mafft))
{
die "Mafft command $cmd_mafft failed";
}
my $fromMAFFT_href = readFASTA($fn_fromMAFFT);
die unless($fromMAFFT_href->{ref});
die unless($fromMAFFT_href->{contig});
print "MAFFT: alignment length went from ", $alignmentLength_before, " to ", length($fromMAFFT_href->{ref}), "\n";
open(OUTPUT, '>', $fn_output_refined) or die;
print OUTPUT $l1, "\n";
print OUTPUT $fromMAFFT_href->{ref}, "\n";
print OUTPUT $fromMAFFT_href->{contig}, "\n";
close(OUTPUT);
}
open(ALIGNMENT, '<', $fn_output) or die "Cannot open $fn_output";
my $l1 = <ALIGNMENT>; chomp($l1);
my $l2 = <ALIGNMENT>; chomp($l2);
my $l3 = <ALIGNMENT>; chomp($l3);
die "Weird first line $l1 in $fn_output" unless($l1 =~ /(\d+) (\d+)-([\-\d]+) (\+|-)(\d+)-(\d+)/);
$score = $1;
$TStart_0based = $2;
$TStop_0based = $3;
$alignmentStrand = $4;
$QStart_0based = $5;
$QStop_0based = $6;
close(ALIGNMENT);
# warn Dumper("Weird strand $alignmentStrand from file $fn_output", $contigID, $overlappingContig_strands{$contigID}, $refContigID) unless($alignmentStrand eq '+');
$T = $l2;
$Q = $l3;
my $Q_noGap = $Q;
my $T_noGap = $T;
$Q_noGap =~ s/-//g;
$T_noGap =~ s/-//g;
if($alignmentStrand eq '+')
{
die Dumper("Alignment contig sequence not equal to original contig sequence", length($Q_noGap), length($contigSequence)) unless(uc($Q_noGap) eq uc($contigSequence));
}
else
{
die Dumper("Alignment contig sequence not equal to original contig sequence", length($Q_noGap), length($contigSequence)) unless(uc($Q_noGap) eq reverseComplement(uc($contigSequence)));
}
die unless(length($T_noGap) == ($TStop_0based - $TStart_0based + 1));
$T_noGap = uc($T_noGap);
my $T_noGaps_equiv = uc(substr($refContigSequence, $TStart_0based, $TStop_0based - $TStart_0based + 1));
for(my $posI = 0; $posI < length($T_noGap); $posI++)
{
my $c1 = substr($T_noGap, $posI, 1);
my $c2 = substr($T_noGaps_equiv, $posI, 1);
next if(($c1 eq 'N') or ($c2 eq 'N'));
die unless($c1 eq $c2);
}
# die Dumper($TStart_0based, $TStop_0based, length($T_noGap), ()) unless(uc($T_noGap) eq uc();
}
die unless(length($T) == length($Q));
die unless((defined $TStart_0based) and (defined $QStart_0based));
die unless($QStart_0based == 0);
die unless($QStop_0based == (length($contigSequence)-1));
$alignments_per_refContig{$refContigID} = [$score, $T, $Q, [$TStart_0based, $TStop_0based], [$QStart_0based, $QStop_0based], $alignmentStrand];
print EDITDISTANCE join("\t", $region, $contigID, $refContigID, $score), "\n";
}
my %alignment_plots_per_refContig;
print "Alignment statistics for $contigID - length ", length($contigSequence), "\n";
foreach my $refContigID (keys %alignments_per_refContig)
{
print " - ", $refContigID, ": strand $alignments_per_refContig{$refContigID}[5], edit distance ", $alignments_per_refContig{$refContigID}[0], "\n";
my $runningPos_T = $alignments_per_refContig{$refContigID}[3][0];
my $runningPos_Q = $alignments_per_refContig{$refContigID}[4][0];
my $L = length($alignments_per_refContig{$refContigID}[1]);
for(my $i = 0; $i < $L; $i++)
{
my $T_c = substr($alignments_per_refContig{$refContigID}[1], $i, 1);
my $Q_c = substr($alignments_per_refContig{$refContigID}[2], $i, 1);
if(($i % 100) == 0)
{
push(@{$alignment_plots_per_refContig{$refContigID}}, [$runningPos_T, $runningPos_Q]);
print DOTPLOTDATA join("\t", $region, $contigID, $refContigID, $runningPos_T, $runningPos_Q), "\n";
}
if(($T_c ne '-'))
{
$runningPos_T++;
}
if(($Q_c ne '-'))
{
$runningPos_Q++;
}
}
}
my @sorted_refContigIDs = sort {$alignments_per_refContig{$a}[0] <=> $alignments_per_refContig{$b}[0]} keys %alignments_per_refContig;
if(scalar(@sorted_refContigIDs) > 1)
{
# die Dumper($sorted_refContigIDs[0], $alignments_per_refContig{$sorted_refContigIDs[0]}[0]);
die unless($alignments_per_refContig{$sorted_refContigIDs[0]}[0] <= $alignments_per_refContig{$sorted_refContigIDs[1]}[0]);
}
foreach my $useIndexForTyping (0, 1, 2)
{
my %extractedSequences;
my %extractedSequences_pos;
my $refContigID = $sorted_refContigIDs[$useIndexForTyping];
print "Extract sequences from alignment to $refContigID\n";
next unless($alignments_per_refContig{$refContigID}[5] eq '+');
my $alignment_running_reference = $alignments_per_refContig{$refContigID}[3][0] - 1;
my $L = length($alignments_per_refContig{$refContigID}[1]);
my $alignment_running_query = -1;
my %found_starts_alignmentCoordinates;
my %found_stops_alignmentCoordinates;
for(my $i = 0; $i < $L; $i++)
{
my $T_c = substr($alignments_per_refContig{$refContigID}[1], $i, 1);
my $Q_c = substr($alignments_per_refContig{$refContigID}[2], $i, 1);
if($T_c ne '-')
{
$alignment_running_reference++;
}
# if($Q_c ne '-')
# {
# die unless($Q_c =~ /^[ACGTN]$/);
# $alignment_running_query++;
# die unless(substr($contigSequence, $alignment_running_query, 1) eq $Q_c);
# }
if($T_c ne '-')
{
if(exists $sequenceExtractionCoordinates_start{$refContigID}{$alignment_running_reference})
{
my @ks = map {join(';-;', @$_)} @{$sequenceExtractionCoordinates_start{$refContigID}{$alignment_running_reference}};
foreach my $k (@ks)
{
die Dumper("Alread found starting coordinates", $k) if(defined $found_starts_alignmentCoordinates{$k});
$found_starts_alignmentCoordinates{$k} = $i;
# die if(defined $found_starts_queryCoordinates{$k});
# $found_starts_queryCoordinates{$k} = $alignment_running_query;
}
}
}
if($T_c ne '-')
{
if(exists $sequenceExtractionCoordinates_stop{$refContigID}{$alignment_running_reference})
{
my @ks = map {join(';-;', @$_)} @{$sequenceExtractionCoordinates_stop{$refContigID}{$alignment_running_reference}};
foreach my $k (@ks)
{
die if(defined $found_stops_alignmentCoordinates{$k});
$found_stops_alignmentCoordinates{$k} = $i;
# die if(defined $found_stops_queryCoordinates{$k});
# $found_stops_queryCoordinates{$k} = $alignment_running_query;
# if(exists $found_starts_alignmentCoordinates{$k})
# {
# my $alignmentSeq = substr($alignments_per_refContig{$refContigID}[2], $found_starts_alignmentCoordinates{$k}, $i - $found_starts_alignmentCoordinates{$k} + 1);
# my $contigSeq = substr($contigSequence, $found_starts_queryCoordinates{$k}, $alignment_running_query - $found_starts_queryCoordinates{$k} + 1);
# print $found_starts_alignmentCoordinates{$k}, " - ", $i, "\n", $found_starts_queryCoordinates{$k}, " - ", $alignment_running_query, "\n", $alignmentSeq, "\n", $contigSeq, "\n\n";
# }
}
}
}
}
my %joint_keys = map {$_ => 1} (keys %found_starts_alignmentCoordinates, keys %found_stops_alignmentCoordinates);
my %positions_per_gene;
my %positions_per_gene_inAlignment;
my %components_per_gene;
foreach my $k (keys %joint_keys)
{
if((exists $found_starts_alignmentCoordinates{$k}) and (exists $found_stops_alignmentCoordinates{$k}))
{
my $l_extract_contig_alignment = $found_stops_alignmentCoordinates{$k} - $found_starts_alignmentCoordinates{$k} + 1;
my $extracted_contig_alignment = substr($alignments_per_refContig{$refContigID}[2], $found_starts_alignmentCoordinates{$k}, $l_extract_contig_alignment);
$extractedSequences{$k} = $extracted_contig_alignment;
my $extracted_contig_alignment_noGap = $extracted_contig_alignment;
$extracted_contig_alignment_noGap =~ s/-//g;
die unless($k =~ /^(.+);-;(.+?)$/);
my $gene = $1;
my $component = $2;
die unless(defined $component);
push(@{$components_per_gene{$gene}}, $component);
#print "EXTRACT $gene $component from $found_starts_alignmentCoordinates{$k} length $l_extract_contig_alignment:\n";
#print substr($alignments_per_refContig{$refContigID}[1], $found_starts_alignmentCoordinates{$k}, $l_extract_contig_alignment), "\n";
#print substr($alignments_per_refContig{$refContigID}[2], $found_starts_alignmentCoordinates{$k}, $l_extract_contig_alignment), "\n";
#print "\n";
die unless (defined $found_starts_alignmentCoordinates{$k});
# my $skipped_query_alignment = substr($alignments_per_refContig{$refContigID}[2], 0, $found_starts_alignmentCoordinates{$k});
# $skipped_query_alignment =~ s/-//g;
# die unless($found_starts_alignmentCoordinates{$k});
# my $total_alignment_till_end_plus_1 = substr($alignments_per_refContig{$refContigID}[2], 0, $found_starts_alignmentCoordinates{$k} + 1);
# $total_alignment_till_end_plus_1 =~ s/-//g;
# $extractedSequences_pos{$k} = [length($skipped_query_alignment), length($total_alignment_till_end_plus_1)];
# note to self: not quite clear what the logic here is - first we set
my $skipped_query_alignment = '';
if($found_starts_alignmentCoordinates{$k})
{
$skipped_query_alignment = substr($alignments_per_refContig{$refContigID}[2], 0, $found_starts_alignmentCoordinates{$k});
}
$skipped_query_alignment =~ s/-//g;
my $first_queryCoordinate = length($skipped_query_alignment);
my $last_queryCoordinate = $first_queryCoordinate + length($extracted_contig_alignment_noGap) - 1;
if(length($extracted_contig_alignment_noGap) == 0)
{
$last_queryCoordinate++;
}
else
{
my $corresponding_raw_contig_sequence = substr($contigSequence, $first_queryCoordinate, $last_queryCoordinate - $first_queryCoordinate + 1);
die Dumper("Mismatch") unless($corresponding_raw_contig_sequence eq $extracted_contig_alignment_noGap);
}
push(@{$positions_per_gene{$gene}}, $first_queryCoordinate, $last_queryCoordinate);
push(@{$positions_per_gene_inAlignment{$gene}}, $found_starts_alignmentCoordinates{$k}, $found_stops_alignmentCoordinates{$k});
}
}
# if(1 == 0)
# {
# foreach my $gene ('HLA-DQB1')
# {
# my @gene_positions = sort @{$positions_per_gene_inAlignment{$gene}};
# my $firstGenePos = $gene_positions[0];
# my $lastGenePos = $gene_positions[$#gene_positions];
# print $gene, " from $firstGenePos length ", $lastGenePos - $firstGenePos + 1, "\n";
# print substr($alignments_per_refContig{$refContigID}[1], $firstGenePos, $lastGenePos - $firstGenePos + 1), "\n";
# print substr($alignments_per_refContig{$refContigID}[2], $firstGenePos, $lastGenePos - $firstGenePos + 1), "\n";
# print substr($alignments_per_refContig{$refContigID}[1], $firstGenePos - 4000, 4000), "\n";
# print substr($alignments_per_refContig{$refContigID}[2], $firstGenePos - 4000, 4000), "\n";
# print substr($alignments_per_refContig{$refContigID}[1], $lastGenePos + 4000, 4000), "\n";
# print substr($alignments_per_refContig{$refContigID}[2], $lastGenePos + 4000, 4000), "\n";
# exit;
# }