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batch_read_mapping_to_reference_genome.pl
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#!/usr/bin/perl
use warnings;
use strict;
use Getopt::Long;
use Env;
use Cwd;
##############################################################
# script: batch_read_mapping_to_reference_genome.pl
# author: Jia-Xing Yue (GitHub ID: yjx1217)
# last edited: 2021.06.17
# description: run batch read mapping to the reference genome
# example: perl batch_read_mapping_to_reference_genome.pl -i Master_Sample_Table.txt -t 4 -o output_dir -reference_genome_assembly_dir ./../01.Reference_Genome_Preprocessing -gamete_reads_dir ./../00.Gamete_Reads -min_mappability 0.85 -window_size 250 -ploidy 1
##############################################################
my $RECOMBINEX_HOME = $ENV{RECOMBINEX_HOME};
my $java_dir = $ENV{java_dir};
my $trimmomatic_dir = $ENV{trimmomatic_dir};
my $bwa_dir = $ENV{bwa_dir};
my $samtools_dir = $ENV{samtools_dir};
my $picard_dir = $ENV{picard_dir};
my $gatk3_dir = $ENV{gatk3_dir};
my $bedtools_dir = $ENV{bedtools_dir};
my $gemtools_dir = $ENV{gemtools_dir};
my $freec_dir = $ENV{freec_dir};
my $sample_table = "Master_Sample_Table.txt";
my $threads = 1;
my $mapping_quality_cutoff_for_mpileup = 30;
my $reference_genome_assembly_dir;
my $gamete_reads_dir;
my $output_dir = "OUTPUT_DIR";
# for aneuploidy detection
my $min_mappability = 0.85;
my $window_size = 250;
my $step_size = 250;
my $ploidy = 1;
my $excluded_chr_list_for_cnv_profiling;
my $debug = "no";
GetOptions('sample_table|i:s' => \$sample_table,
'threads|t:i' => \$threads,
'mapping_quality_cutoff_for_mpileup|q:i' => \$mapping_quality_cutoff_for_mpileup,
'reference_genome_assembly_dir|rga_dir:s' => \$reference_genome_assembly_dir,
'gamete_reads_dir|or_dir:s' => \$gamete_reads_dir,
'output_dir|o_dir:s' => \$output_dir,
'min_mappability|min_map:f' => \$min_mappability,
'window|w:i' => \$window_size,
'step|s:i' => \$step_size,
'ploidy|p:i' => \$ploidy,
'excluded_chr_list_for_cnv_profiling|e:s' => \$excluded_chr_list_for_cnv_profiling,
'debug|d:s' => \$debug);
my $sample_table_fh = read_file($sample_table);
my %sample_table = ();
my @sample_table = ();
parse_sample_table_by_spore($sample_table_fh, \%sample_table, \@sample_table);
my $base_dir = cwd();
system("mkdir $output_dir");
my $all_samples_cnv = "$base_dir/$output_dir/all_samples.CNV_summary.txt";
my $all_samples_cnv_fh = write_file($all_samples_cnv);
print $all_samples_cnv_fh "sample\tref_genome\tchr\tstart\tend\tcopy_number\tstatus\tMWU_test_p_value\tKS_test_p_value\n";
my $sample_count = 0;
my $mapping_count = 0;
my $adapter = "$trimmomatic_dir/adapters/TruSeq3-PE-2.fa";
foreach my $sample_id (@sample_table) {
my $local_time = localtime();
print "[$local_time] processing sample $sample_id with short-read mapping\n";
$sample_count++;
my $R1_read_file = "$base_dir/$gamete_reads_dir/$sample_table{$sample_id}{'R1_read_file'}";
my $R2_read_file = "$base_dir/$gamete_reads_dir/$sample_table{$sample_id}{'R2_read_file'}";
print "processing sample $sample_id\n";
print "PE_reads_file = $R1_read_file,$R2_read_file\n";
print "Check the specified short read file:\n";
if (-e $R1_read_file) {
print "Successfully located the specified short read file 1: $R1_read_file.\n";
} else {
print "Cannot find the specified short read file 1: $R1_read_file!\n";
print "Exit!\n";
exit;
}
if (-e $R2_read_file) {
print "Successfully located the specified short read file 2: $R2_read_file.\n";
} else {
print "Cannot find the specified short read file 2: $R2_read_file!\n";
print "Exit!\n";
exit;
}
my $excluded_chr_list_for_cnv_profiling_file = "$base_dir/$excluded_chr_list_for_cnv_profiling";
print "Check the specified excluded_chr_list_for_cnv_profiling file:\n";
if (-e $excluded_chr_list_for_cnv_profiling_file) {
print "Successfully located the specified excluded_chr_list_for_cnv_profiling_file: $excluded_chr_list_for_cnv_profiling_file.\n";
} else {
print "Cannot find the specified excluded_chr_list_for_cnv_profiling_file: $excluded_chr_list_for_cnv_profiling_file!\n";
print "Exit!\n";
exit;
}
# my $R1_read_fh = read_file($R1_read_file);
# my $raw_read_length = get_read_length($R1_read_fh);
my $raw_read_length = 100;
print "raw read length = $raw_read_length\n";
my $cross_pair = $sample_table{$sample_id}{'cross_pair'};
my $tetrad_id = $sample_table{$sample_id}{'tetrad_id'};
my $spore_id = $sample_table{$sample_id}{'spore_id'};
my $sample_tag = "$cross_pair.$tetrad_id.$spore_id";
my ($genome1_tag, $genome2_tag) = split /-/, $cross_pair;
print "genome1: $genome1_tag, genome2: $genome2_tag\n";
# my @parent_genome_tags = ($genome1_tag, $genome2_tag);
my @reference_genome_tag = ("ref");
my $sample_output_dir;
foreach my $reference_genome_tag (@reference_genome_tag) {
print "mapping to $reference_genome_tag\n";
$mapping_count++;
my $reference_genome_assembly = "$base_dir/$reference_genome_assembly_dir/${reference_genome_tag}.genome.raw.relabel.fa";
if (-e $reference_genome_assembly) {
print "Successfully located the expected reference_genome_assembly file: $reference_genome_assembly.\n";
} else {
print "Cannot find the expected reference genome assembly file: $reference_genome_assembly!\n";
print "Exit!\n";
exit;
}
$sample_output_dir = "$base_dir/$output_dir/${sample_tag}.${reference_genome_tag}";
system("mkdir -p $sample_output_dir");
chdir("$sample_output_dir") or die "cannot change directory to: $!\n";
system("mkdir tmp");
print "trim the reads by trimmomatic\n";
system("ln -s $adapter adapter.fa");
system("ln -s $R1_read_file $sample_tag.R1.raw.fq.gz");
system("ln -s $R2_read_file $sample_tag.R2.raw.fq.gz");
system("$java_dir/java -Djava.io.tmpdir=./tmp -XX:ParallelGCThreads=$threads -jar $trimmomatic_dir/trimmomatic.jar PE -threads $threads -phred33 $sample_tag.R1.raw.fq.gz $sample_tag.R2.raw.fq.gz $sample_tag.R1.trimmed.PE.fq.gz $sample_tag.R1.trimmed.SE.fq.gz $sample_tag.R2.trimmed.PE.fq.gz $sample_tag.R2.trimmed.SE.fq.gz ILLUMINACLIP:adapter.fa:2:30:10 SLIDINGWINDOW:5:20 MINLEN:36");
if ($debug eq "no") {
system("rm $sample_tag.R1.raw.fq.gz");
system("rm $sample_tag.R2.raw.fq.gz");
system("rm $sample_tag.R1.trimmed.SE.fq.gz");
system("rm $sample_tag.R2.trimmed.SE.fq.gz");
system("rm adapter.fa");
}
print("mapping the reads by bwa\n");
system("ln -s $reference_genome_assembly $reference_genome_tag.genome.fa");
system("$bwa_dir/bwa index $reference_genome_tag.genome.fa");
system("$bwa_dir/bwa mem -t $threads -M $reference_genome_tag.genome.fa $sample_tag.R1.trimmed.PE.fq.gz $sample_tag.R2.trimmed.PE.fq.gz | $samtools_dir/samtools view -bS -q $mapping_quality_cutoff_for_mpileup -F 3340 -f 2 - >${sample_tag}.${reference_genome_tag}.bam");
if ($debug eq "no") {
system("rm $reference_genome_tag.genome.fa.bwt");
system("rm $reference_genome_tag.genome.fa.pac");
system("rm $reference_genome_tag.genome.fa.ann");
system("rm $reference_genome_tag.genome.fa.amb");
system("rm $reference_genome_tag.genome.fa.sa");
system("rm $sample_tag.R1.trimmed.PE.fq.gz");
system("rm $sample_tag.R2.trimmed.PE.fq.gz");
}
## index reference sequence
system("$samtools_dir/samtools faidx $reference_genome_tag.genome.fa");
system("$java_dir/java -Djava.io.tmpdir=./tmp -Dpicard.useLegacyParser=false -XX:ParallelGCThreads=$threads -jar $picard_dir/picard.jar CreateSequenceDictionary -REFERENCE $reference_genome_tag.genome.fa -OUTPUT $reference_genome_tag.genome.dict");
## sort bam file by picard-tools: SortSam
system("$java_dir/java -Djava.io.tmpdir=./tmp -Dpicard.useLegacyParser=false -XX:ParallelGCThreads=$threads -jar $picard_dir/picard.jar SortSam -INPUT ${sample_tag}.${reference_genome_tag}.bam -OUTPUT ${sample_tag}.${reference_genome_tag}.sort.bam -SORT_ORDER coordinate -MAX_RECORDS_IN_RAM 1000000");
if ($debug eq "no") {
system("rm ${sample_tag}.${reference_genome_tag}.bam");
}
## fixmate
system("$java_dir/java -Djava.io.tmpdir=./tmp -Dpicard.useLegacyParser=false -XX:ParallelGCThreads=$threads -jar $picard_dir/picard.jar FixMateInformation -INPUT ${sample_tag}.${reference_genome_tag}.sort.bam -OUTPUT ${sample_tag}.${reference_genome_tag}.fixmate.bam");
if ($debug eq "no") {
system("rm ${sample_tag}.${reference_genome_tag}.sort.bam");
}
## add or replace read groups and sort
system("$java_dir/java -Djava.io.tmpdir=./tmp -Dpicard.useLegacyParser=false -XX:ParallelGCThreads=$threads -jar $picard_dir/picard.jar AddOrReplaceReadGroups -INPUT=${sample_tag}.${reference_genome_tag}.fixmate.bam -OUTPUT ${sample_tag}.${reference_genome_tag}.rdgrp.bam -SORT_ORDER coordinate -MAX_RECORDS_IN_RAM 1000000 -RGID ${sample_tag}.${reference_genome_tag} -RGLB ${sample_tag}.${reference_genome_tag} -RGPL 'Illumina' -RGPU ${sample_tag}.${reference_genome_tag} -RGSM ${sample_tag} -RGCN 'RGCN'");
if ($debug eq "no") {
system("rm ${sample_tag}.${reference_genome_tag}.fixmate.bam");
}
# Picard tools remove duplicates
system("$java_dir/java -Djava.io.tmpdir=./tmp -Dpicard.useLegacyParser=false -XX:ParallelGCThreads=$threads -jar $picard_dir/picard.jar MarkDuplicates -INPUT ${sample_tag}.${reference_genome_tag}.rdgrp.bam -MAX_FILE_HANDLES_FOR_READ_ENDS_MAP 1000 -REMOVE_DUPLICATES true -METRICS_FILE ${sample_tag}.${reference_genome_tag}.dedup.matrics -OUTPUT ${sample_tag}.${reference_genome_tag}.dedup.bam");
# index the dedup.bam file
system("$samtools_dir/samtools index ${sample_tag}.${reference_genome_tag}.dedup.bam");
if ($debug eq "no") {
system("rm ${sample_tag}.${reference_genome_tag}.rdgrp.bam");
}
# GATK local realign
# find realigner targets
system("$java_dir/java -Djava.io.tmpdir=./tmp -XX:ParallelGCThreads=$threads -jar $gatk3_dir/gatk3.jar -nt $threads -R $reference_genome_tag.genome.fa -T RealignerTargetCreator -I ${sample_tag}.${reference_genome_tag}.dedup.bam -o ${sample_tag}.${reference_genome_tag}.realn.intervals");
# run realigner
system("$java_dir/java -Djava.io.tmpdir=./tmp -XX:ParallelGCThreads=$threads -jar $gatk3_dir/gatk3.jar -R $reference_genome_tag.genome.fa -T IndelRealigner -I ${sample_tag}.${reference_genome_tag}.dedup.bam -targetIntervals ${sample_tag}.${reference_genome_tag}.realn.intervals -o ${sample_tag}.${reference_genome_tag}.realn.bam");
# index final bam file
system("$samtools_dir/samtools index ${sample_tag}.${reference_genome_tag}.realn.bam");
if ($debug eq "no") {
system("rm ${sample_tag}.${reference_genome_tag}.dedup.bam");
system("rm ${sample_tag}.${reference_genome_tag}.dedup.bam.bai");
system("rm ${sample_tag}.${reference_genome_tag}.dedup.matrics");
system("rm ${sample_tag}.${reference_genome_tag}.realn.intervals");
}
# generate samtools mpileup
system("$samtools_dir/samtools mpileup -Q 0 -C 0 -q $mapping_quality_cutoff_for_mpileup -f $reference_genome_tag.genome.fa ${sample_tag}.${reference_genome_tag}.realn.bam |gzip -c >${sample_tag}.${reference_genome_tag}.mpileup.gz");
# compute basic alignment statistics by samtools
system("$samtools_dir/samtools flagstat ${sample_tag}.${reference_genome_tag}.realn.bam >${sample_tag}.${reference_genome_tag}.samstat");
# calculate per-base depth
system("$samtools_dir/samtools depth -aa ${sample_tag}.${reference_genome_tag}.realn.bam |gzip -c >${sample_tag}.${reference_genome_tag}.depth.txt.gz");
# compute insert size statistics
# system("$java_dir/java -Djava.io.tmpdir=./tmp -Dpicard.useLegacyParser=false -XX:ParallelGCThreads=$threads -jar $picard_dir/picard.jar CollectInsertSizeMetrics -I ${sample_tag}.${reference_genome_tag}.realn.bam -O ${sample_tag}.${reference_genome_tag}.insert_size_metrics.txt -H ${sample_tag}.${reference_genome_tag}.insert_size_histogram.pdf -M 0.5");
# calculate read mapping coverage statistics
system("perl $RECOMBINEX_HOME/scripts/summarize_mapping_coverage.pl -r $reference_genome_tag.genome.fa -s ${sample_tag}.${reference_genome_tag}.samstat -d ${sample_tag}.${reference_genome_tag}.depth.txt.gz -c 5 -t $sample_id -o ${sample_tag}.${reference_genome_tag}.coverage_summary.txt");
if ($mapping_count == 1) {
system("cp ${sample_tag}.${reference_genome_tag}.coverage_summary.txt ./../all_samples.coverage_summary.txt");
} else {
system("tail -1 ${sample_tag}.${reference_genome_tag}.coverage_summary.txt >> ./../all_samples.coverage_summary.txt");
}
if ($debug eq "no") {
system("rm ${sample_tag}.${reference_genome_tag}.depth.txt.gz");
}
# scan for aneuploidy with FREEC
my $gc_range_file = "$base_dir/$reference_genome_assembly_dir/ref.FREEC.GC_range.txt";
my $gc_range_fh = read_file($gc_range_file);
my ($min_expected_gc, $max_expected_gc) = extract_gc_range($gc_range_fh);
system("perl $RECOMBINEX_HOME/scripts/run_FREEC_wrapper_lite_for_RecombineX.pl -r $reference_genome_tag -bam ${sample_tag}.${reference_genome_tag}.realn.bam -prefix ${sample_tag}.${reference_genome_tag} -ploidy $ploidy -bedtools $bedtools_dir/bedtools -samtools $samtools_dir/samtools -freec $freec_dir/freec -window $window_size -step $step_size -read_length_for_mappability $raw_read_length -min_mappability $min_mappability -min_expected_gc $min_expected_gc -max_expected_gc $max_expected_gc -mates_orientation 0 -threads $threads -refseq_genome_preprocessing_dir ./../../$reference_genome_assembly_dir -excluded_chr_list ./../../$excluded_chr_list_for_cnv_profiling");
system("Rscript --vanilla --slave $RECOMBINEX_HOME/scripts/CNV_segmentation_by_DNAcopy.R --input ${sample_tag}.${reference_genome_tag}.FREEC.bam_ratio.txt --prefix ${sample_tag}.${reference_genome_tag} --window $window_size --ploidy $ploidy --genome_fai $reference_genome_tag.genome.fa.fai");
system("perl $RECOMBINEX_HOME/scripts/adjust_FREEC_copynumber_by_DNAcopy_copynumber.pl -i ${sample_tag}.${reference_genome_tag}.FREEC.bam_ratio.sorted.txt -a ${sample_tag}.${reference_genome_tag}.FREEC.bam_ratio.sorted.resegmented.lite.txt -o ${sample_tag}.${reference_genome_tag}.FREEC.bam_ratio.sorted.adjusted.txt");
if ( -s "${sample_tag}.${reference_genome_tag}.FREEC.bam_ratio.sorted.adjusted.txt") {
system("Rscript --vanilla --slave $RECOMBINEX_HOME/scripts/plot_CNV_for_FREEC.R --ploidy $ploidy --genome_fai $reference_genome_tag.genome.fa.fai --input ${sample_tag}.${reference_genome_tag}.FREEC.bam_ratio.sorted.adjusted.txt --output ${sample_tag}.${reference_genome_tag}.CNV_plot.pdf");
system("rm Rplots.pdf");
if ( -s "${sample_tag}.${reference_genome_tag}.FREEC.bam_ratio.sorted.resegmented.CNVs.txt") {
system("Rscript --vanilla --slave $RECOMBINEX_HOME/scripts/assess_CNV_significance_for_FREEC.R --cnv ${sample_tag}.${reference_genome_tag}.FREEC.bam_ratio.sorted.resegmented.CNVs.txt --ratio ${sample_tag}.${reference_genome_tag}.FREEC.bam_ratio.sorted.adjusted.txt --genome_fai $reference_genome_tag.genome.fa.fai --output ${sample_tag}.${reference_genome_tag}.CNV_significance_test.txt");
my $sample_cnv_fh = read_file("${sample_tag}.${reference_genome_tag}.CNV_significance_test.txt");
parse_sample_cnv_file($sample_cnv_fh, $sample_id, "$reference_genome_tag.genome.fa", $all_samples_cnv_fh);
close $sample_cnv_fh;
} else {
system("echo -e \"chr\tstart\tend\tcopy_number\tstatus\tMWU_test_p_value\tKS_test_p_value\" > ${sample_tag}.${reference_genome_tag}.CNV_significance_test.txt");
}
} else {
system(" echo \"Exception encountered for FREEC! Exit! ...\" > ${sample_tag}.${reference_genome_tag}.realn.bam.no_FREEC.txt");
}
if ($debug eq "no") {
# remove old files
system("rm for_CNV.bam");
system("rm FREEC.bam");
system("rm FREEC.bam.header.old.sam");
system("rm FREEC.bam.header.new.sam");
system("rm GC_profile.${window_size}bp.cnp");
system("rm -r tmp");
}
$local_time = localtime();
print "[$local_time] finishing short-read mapping for sample $sample_id \n";
chdir("./../") or die "cannot change directory to: $!\n";
}
}
close $all_samples_cnv_fh;
print "A total of $sample_count samples were processed!\n";
sub read_file {
my $file = shift @_;
my $fh;
if ($file =~ /\.gz$/) {
open($fh, "gunzip -c $file |") or die "can't open pipe to $file";
}
else {
open($fh, $file) or die "can't open $file";
}
return $fh;
}
sub write_file {
my $file = shift @_;
my $fh;
if ($file =~ /\.gz$/) {
open($fh, "| gzip -c >$file") or die "can't open $file\n";
} else {
open($fh, ">$file") or die "can't open $file\n";
}
return $fh;
}
sub parse_sample_table_by_spore {
my ($fh, $sample_table_hashref, $sample_table_arrayref) = @_;
while (<$fh>) {
chomp;
/^#/ and next;
/^\s*$/ and next;
my ($sample_id, $tetrad_id, $spore_id, $PE_reads_files, $cross_pair, $note) = split /\s+/, $_;
push @$sample_table_arrayref, $sample_id;
my ($R1_read_file, $R2_read_file) = split /,/, $PE_reads_files;
$$sample_table_hashref{$sample_id}{'tetrad_id'} = $tetrad_id;
$$sample_table_hashref{$sample_id}{'spore_id'} = $spore_id;
$$sample_table_hashref{$sample_id}{'R1_read_file'} = $R1_read_file;
$$sample_table_hashref{$sample_id}{'R2_read_file'} = $R2_read_file;
$$sample_table_hashref{$sample_id}{'cross_pair'} = $cross_pair;
$$sample_table_hashref{$sample_id}{'note'} = $note;
}
}
sub parse_sample_cnv_file {
my ($sample_cnv_fh, $sample, $refseq, $all_samples_cnv_fh) = @_;
while (<$sample_cnv_fh>) {
chomp;
/^#/ and next;
/^\s*$/ and next;
/chr\tstart/ and next;
my ($chr, $start, $end, $copy_number, $status, $MWU_test_p_value, $KS_test_p_value) = split /\t/, $_;
# if ($MWU_test_p_value < 0.05) {
print $all_samples_cnv_fh "$sample\t$refseq\t$_\n";
#}
}
}
sub get_read_length {
my $fh = shift @_;
my $read_length = 100;
while (<$fh>) {
chomp;
if ($. % 4 == 2) {
$read_length = length $_;
last;
}
}
return $read_length;
}
sub extract_gc_range {
my $fh = shift @_;
my $min_expected_gc;
my $max_expected_gc;
while (<$fh>) {
chomp;
/^#/ and next;
/^\s*$/ and next;
($min_expected_gc, $max_expected_gc) = split /\t/, $_;
}
return ($min_expected_gc, $max_expected_gc);
}