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pughlab_fragmentomics_pipeline.pl
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pughlab_fragmentomics_pipeline.pl
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#!/usr/bin/env perl
### pughlab_fragmentomics_pipeline.pl ##############################################################
use AutoLoader 'AUTOLOAD';
use strict;
use warnings;
use Carp;
use Getopt::Std;
use Getopt::Long;
use POSIX qw(strftime);
use File::Basename;
use File::Path qw(make_path);
use YAML qw(LoadFile);
use List::Util qw(any);
use File::Find;
use Data::Dumper;
use IO::Handle;
my $cwd = dirname(__FILE__);
require "$cwd/scripts/utilities.pl";
# define path to fragmentomics code location(s)
our ($fragmentomics_code_dir, $griffin_code_dir) = undef;
# define some global variables
our ($reference, $reference_peaks) = undef;
our ($delfi_healthy, $delfi_gaps, $delfi_vntrs, $delfi_tiles, $delfi_filters) = undef;
our ($tfbs_sites, $tcga_sites, $dhs_sites, $hk_sites) = undef;
our $griffin_sites;
our ($griffin_mappable, $griffin_mappable_name) = undef;
####################################################################################################
# version author comment
# 1.0 sprokopec wrapper to run Derek's fragmentomics code
### DEFINE SUBROUTINES #############################################################################
# run downsample bam
sub create_downsample_command {
my %args = (
bam => undef,
id => undef,
n_reads => undef,
factor => undef,
outdir => undef,
tmpdir => undef,
@_
);
my $downsample_command;
if (defined($args{n_reads})) {
$downsample_command = join("\n",
"N_READS=" . $args{n_reads},
"TOTAL_READS=\$(samtools view -c $args{bam})",
"SCALE_FACTOR=\$(printf '%.4f\\n' \$(echo \"\$N_READS/\$TOTAL_READS\" | bc -l))"
);
} elsif (defined($args{factor})) {
$downsample_command = join("\n",
"SCALE_FACTOR=$args{factor}",
"TOTAL_READS=\$(samtools view -c $args{bam})",
"N_READS=\$(printf '%.0f\\n' \$(echo \"\$SCALE_FACTOR*\$TOTAL_READS\" | bc -l))"
);
}
$downsample_command .= "\n" . join("\n",
'echo "downsampling to $N_READS from $TOTAL_READS (scale factor = $SCALE_FACTOR)"' . "\n",
'if (( $(echo "$SCALE_FACTOR < 1" | bc -l) )); then'
);
$downsample_command .= "\n " . join(' ',
'java -Xmx1g',
'-Djava.io.tmpdir=' . $args{tmpdir},
'-jar $picard_dir/picard.jar DownsampleSam',
'I=' . $args{bam},
'O=' . $args{outdir} . '/' . $args{id} . '_downsampled.bam',
'P=$SCALE_FACTOR',
'VALIDATION_STRINGENCY=LENIENT;'
);
$downsample_command .= "\nelse\n " . join(' ',
'ln -s',
$args{bam},
$args{outdir} . '/' . $args{id} . '_downsampled.bam;'
);
$downsample_command .= "\nfi";
$downsample_command .= "\n" . join(' ',
'samtools index',
$args{outdir} . '/' . $args{id} . '_downsampled.bam;'
);
return($downsample_command);
}
# run DELFI fragmentomics ratio
sub create_ratio_command {
my %args = (
bam => undef,
id => undef,
outdir => undef,
@_
);
my $delfi_command = join(' ',
"Rscript $fragmentomics_code_dir/ratio/runFrag.R",
"--id", $args{id},
"--bam", $args{bam},
"--outdir", $args{outdir},
"--libdir $fragmentomics_code_dir/ratio",
"--filters $delfi_filters",
"--gaps $delfi_gaps",
"--VNTRs $delfi_vntrs",
"--tiles $delfi_tiles",
"--healthy $delfi_healthy"
);
return($delfi_command);
}
# run VESSIES fragment score
sub create_fragmentscore_command {
my %args = (
id => undef,
bam => undef,
outdir => undef,
@_
);
my $score_command = join(' ',
"Rscript $fragmentomics_code_dir/fragment_score/scripts/04_patient_score.R",
"--id", $args{id},
"--bam", $args{bam},
"--outdir", $args{outdir},
"--ref $fragmentomics_code_dir/fragment_score/ref/vessies_reference_set.txt",
"--libdir $fragmentomics_code_dir/fragment_score/scripts"
);
return($score_command);
}
# format command to create de-duplicated BAM
sub get_deduplicate_command {
my %args = (
id => undef,
bam => undef,
outdir => undef,
tmp_dir => undef,
java_mem => undef,
dedup_tool => 'picard',
@_
);
my $output_stem = join('/', $args{outdir}, $args{id});
# deduplicate BAM
my $dedup_command = "# deduplicate bam";
if ('picard' eq $args{dedup_tool}) {
$dedup_command .= "\n" . join(' ',
'java -Xmx' . $args{java_mem},
'-Djava.io.tmpdir=' . $args{tmp_dir},
'-jar $picard_dir/picard.jar MarkDuplicates',
'I=' . $args{bam},
'O=' . $output_stem . '_deduped.bam',
'M=' . $output_stem . '_deduped.metrics',
'REMOVE_DUPLICATES=true',
'REMOVE_SEQUENCING_DUPLICATES=true'
);
} else {
$dedup_command .= "\n" . join(' ',
'sambamba markdup',
'-t 1 -r',
$args{bam},
$output_stem . '_deduped.bam',
'--tmpdir=' . $args{tmp_dir},
'--overflow-list-size 5000000'
);
}
# sort deduplicated BAM
$dedup_command .= "\n\n# sort deduplicated bam";
$dedup_command .= "\n" . join(' ',
'samtools sort -n',
$output_stem . '_deduped.bam',
'-o', $output_stem . '_deduped_sorted.bam'
);
# convert to bedpe (all reads)
$dedup_command .= "\n\n# convert deduplicated bam to bedpe";
$dedup_command .= "\n" . join(' ',
'samtools view -bf 0x2',
$output_stem . '_deduped_sorted.bam',
'| bedtools bamtobed -i stdin -bedpe',
'>', $output_stem . '_all.bedpe'
);
# convert to bedpe (high quality reads)
$dedup_command .= "\n\n# select high-quality reads and convert to bedpe";
$dedup_command .= "\n" . join(' ',
'samtools view -bf 0x2 -q 30',
$output_stem . '_deduped_sorted.bam',
'| bedtools bamtobed -i stdin -bedpe',
'>', $output_stem . '_q30.bedpe'
);
# remove intermediates
$dedup_command .= "\n\n# check for completeness and remove intermediates";
$dedup_command .= "\n" . join("\n",
'if [[ -f ' . $output_stem . '_q30.bedpe ]]; then',
' rm ' . $output_stem . '*deduped*',
'fi'
);
return($dedup_command);
}
# format command to create size-selected and de-duplicated BAM
sub get_sized_deduplicate_command {
my %args = (
id => undef,
bam => undef,
outdir => undef,
size => 'genome',
tmp_dir => undef,
java_mem => undef,
@_
);
my $output_stem = join('/', $args{outdir}, $args{id});
my ($dedup_command, $new_bam);
# select fragments of 167bp length (ie, 'normal' cfDNA)
$dedup_command = "# extracting desired fragments: ($args{size})";
if ('genome' eq $args{size}) {
$new_bam = $output_stem . "_len167_sorted.bam";
$output_stem = join('/', $args{outdir}, $args{id} . '_len167');
$dedup_command .= "\n" . join(' ',
"samtools view -h $args{bam}",
"| awk 'substr(\$0,1,1)==\"@\" || (\$9==167) || (\$9==-167)'",
"| samtools view -b",
"> $output_stem.bam;"
);
# select fragments of 150bp length (ie, 'short' cfDNA)
} elsif ('short' eq $args{size}) {
$new_bam = $output_stem . "_len150_sorted.bam";
$output_stem = join('/', $args{outdir}, $args{id} . '_len150');
$dedup_command .= "\n" . join(' ',
"samtools view -h $args{bam}",
"| awk 'substr(\$0,1,1)==\"@\" || (\$9<=150 && \$9>=10) || (\$9>=-150 && \$9<=-10)'",
"| samtools view -b",
"> $output_stem.bam;"
);
}
$dedup_command .= "\n\n# sort and index size-selected bam";
$dedup_command .= "\nsamtools index $output_stem.bam";
$dedup_command .= "\nsamtools sort $output_stem.bam -o $output_stem\_sorted.bam";
$dedup_command .= "\nsamtools index $output_stem\_sorted.bam";
# deduplicate BAM
$dedup_command .= "\n\n# deduplicate size-selected bam";
$dedup_command .= "\n" . join(' ',
'java -Xmx' . $args{java_mem},
'-Djava.io.tmpdir=' . $args{tmp_dir},
'-jar $picard_dir/picard.jar MarkDuplicates',
'I=' . $new_bam,
'O=' . $output_stem . '_deduped.bam',
'M=' . $output_stem . '_deduped.metrics',
'REMOVE_DUPLICATES=true',
'REMOVE_SEQUENCING_DUPLICATES=true'
);
# sort deduplicated BAM
$dedup_command .= "\n\n# sort deduplicated size-selected bam";
$dedup_command .= "\n" . join(' ',
'samtools sort -n',
$output_stem . '_deduped.bam',
'-o', $output_stem . '_deduped_sorted.bam'
);
# convert to bedpe (all reads)
$dedup_command .= "\n\n# convert bam to bedpe";
$dedup_command .= "\n" . join(' ',
'samtools view -bf 0x2',
$output_stem . '_deduped_sorted.bam',
'| bedtools bamtobed -i stdin -bedpe',
'>', $output_stem . '.bedpe'
);
# remove intermediates
$dedup_command .= "\n\n# check for completeness and remove intermediates";
$dedup_command .= "\n" . join("\n",
"if [[ -f $output_stem.bedpe ]]; then",
' rm ' . $output_stem . '*bam*',
' rm ' . $output_stem . '*metrics',
'fi'
);
return($dedup_command);
}
# run nucleosome positioning
sub create_nucleosome_position_command {
my %args = (
id => undef,
bedpe => undef,
outdir => undef,
@_
);
my $output_stem = join('/', $args{outdir}, $args{id});
my $linked_bedpe = join('/', $args{outdir}, $args{id} . '.bedpe' );
my $nuc_pos_command = "ln -s $args{bedpe} $linked_bedpe";
$nuc_pos_command .= "\n\n# split bedpe by chromosome";
$nuc_pos_command .= "\necho '>>> Running splitBedpe.sh <<<'";
$nuc_pos_command .= "\n\n" . join(' ',
"$fragmentomics_code_dir/nucleosome_peak/scripts/splitBedpe.sh",
$linked_bedpe
);
$nuc_pos_command .= "\n\n# calculate distance to nucleosome peaks";
$nuc_pos_command .= "\necho '>>> Running nucleosome_peaks_distance.R <<<'";
$nuc_pos_command .= "\n\n" . join(' ',
"Rscript $fragmentomics_code_dir/nucleosome_peak/scripts/nucleosome_peaks_distance.R",
"--id", $args{id},
"--path", $args{outdir},
"--peaks", $reference_peaks,
"--outdir", $args{outdir}
);
$nuc_pos_command .= "\n\n# check for completeness and remove intermediates";
$nuc_pos_command .= "\n" . join("\n",
'if [[ -f ' . $output_stem . '_peak_distance.txt ]]; then',
" echo '>>> Nucleosome Position Profiling completed successfully! <<<'",
' rm ' . $output_stem . '*.bed*',
'fi'
);
return($nuc_pos_command);
}
# format command to extract insert size metrics
sub get_insert_sizes_command {
my %args = (
id => undef,
bam => undef,
outdir => undef,
java_mem => undef,
tmp_dir => undef,
@_
);
my $output_stem = join('/', $args{outdir}, $args{id});
my $fs_command = join(' ',
'java -Xmx' . $args{java_mem},
'-Djava.io.tmpdir=' . $args{tmp_dir},
'-jar $picard_dir/picard.jar CollectInsertSizeMetrics',
'I=' . $args{bam},
'O=' . $output_stem . '_picard.txt',
'H=' . $output_stem . '.pdf',
'M=0 W=600'
);
return($fs_command);
}
# format command to run Griffin GC functions
sub get_griffin_gc_command {
my %args = (
id => undef,
bam => undef,
outdir => undef,
tmpdir => undef,
n_cpus => 8,
@_
);
my $gc_command;
# using Griffin v0.1.0 (version provided with Pipeline-Suite)
unless ($griffin_code_dir =~ m/v0.2.0/) {
$gc_command = join(' ',
"$griffin_code_dir/scripts/griffin_GC_counts.py",
'--bam_file', $args{bam},
'--bam_file_name', $args{id},
'--mapable_regions', $griffin_mappable,
'--ref_seq', $reference,
'--chrom_sizes', "$griffin_code_dir/Ref/hg38.standard.chrom.sizes",
'--out_dir', $args{outdir},
'--map_q 20 --size_range 15 500 --CPU', $args{n_cpus}
);
$gc_command .= "\n\n" . join(' ',
"$griffin_code_dir/scripts/griffin_GC_bias.py",
'--bam_file_name', $args{id},
'--mapable_name', $griffin_mappable_name,
'--genome_GC_frequency', "$griffin_code_dir/Ref/genome_GC_frequency",
'--out_dir', $args{outdir},
'--size_range 15 500'
);
} else {
# using Griffin v0.2.0 (currently doesn't work; testing in progress)
$gc_command = "source activate base";
$gc_command .= "\nconda activate griffin2";
my $map_stem = join('/', $args{outdir}, 'mappability_bias', $args{id} . '.mappability_bias');
$gc_command .= "\n\n" . join(' ',
"$griffin_code_dir/scripts/griffin_mappability_correction.py",
'--bam_file', $args{bam},
'--bam_file_name', $args{id},
'--output', $map_stem . '.txt',
'--output_plot', $map_stem . '.pdf',
'--mappability', "$griffin_code_dir/Ref/k50.Umap.MultiTrackMappability.hg38.bw",
'--exclude_paths', "$griffin_code_dir/Ref/encode_unified_GRCh38_exclusion_list.bed",
'--chrom_sizes', "$griffin_code_dir/Ref/hg38.standard.chrom.sizes",
'--tmp_dir', $args{tmpdir},
'--map_quality 20 --CPU', $args{n_cpus}
);
$gc_command .= "\n\n" . join(' ',
"$griffin_code_dir/scripts/griffin_GC_counts.py",
'--bam_file', $args{bam},
'--bam_file_name', $args{id},
'--mappable_regions_path', "$griffin_code_dir/Ref/k100_minus_exclusion_lists.mappable_regions.hg38.bed",
'--ref_seq', $reference,
'--chrom_sizes', "$griffin_code_dir/Ref/hg38.standard.chrom.sizes",
'--out_dir', $args{outdir},
'--map_q 20 --size_range 15 500 --CPU', $args{n_cpus}
);
$gc_command .= "\n\n" . join(' ',
"$griffin_code_dir/scripts/griffin_GC_bias.py",
'--bam_file_name', $args{id},
'--mappable_name k100_minus_exclusion_lists.mappable_regions.hg38',
'--genome_GC_frequency', "$griffin_code_dir/Ref/genome_GC_frequency",
'--out_dir', $args{outdir},
'--size_range 15 500'
);
}
return($gc_command);
}
# format command to run Griffin nucleosome profiling
sub get_griffin_profiling_command {
my %args = (
id => undef,
bam => undef,
gc_bias => undef,
map_bias => undef,
n_cpus => 8,
tmpdir => undef,
outdir => undef,
sites => undef,
@_
);
my $griffin_command;
# using Griffin v0.1.0 (version provided with Pipeline-Suite)
unless ($griffin_code_dir =~ m/v0.2.0/) {
$griffin_command = join(' ',
"$griffin_code_dir/scripts/griffin_calc_coverage.py",
'--sample_name', $args{id},
'--bam', $args{bam},
'--reference_genome', $reference,
'--GC_bias', $args{gc_bias},
'--background_normalization None',
'--sites_yaml', $args{sites},
'--results_dir', $args{outdir},
'--chrom_column Chrom',
'--strand_column Strand',
'--chroms chr1 chr2 chr3 chr4 chr5 chr6 chr7 chr8 chr9 chr10 chr11 chr12 chr13 chr14 chr15 chr16 chr17 chr18 chr19 chr20 chr21 chr22',
'--norm_window -5000 5000',
'--plot_window -1000 1000',
'--fragment_length 165',
'--step 15 --size_range 90 220 --map_quality 20',
'--individual False --smoothing True --num_sites none',
'--sort_by none --ascending none --cpu', $args{n_cpus},
'--erase_intermediates False'
);
} else {
# using Griffin v0.2.0 (currently doesn't work; testing in progress)
$griffin_command = "source activate base";
$griffin_command .= "\nconda activate griffin2";
$griffin_command .= "\n\n" . join(' ',
"$griffin_code_dir/scripts/griffin_coverage.py",
'--sample_name', $args{id},
'--bam', $args{bam},
'--reference_genome', $reference,
'--GC_bias', $args{gc_bias},
'--mappability_bias', $args{map_bias},
'--mappability_correction True',
'--mappability_bw', "$griffin_code_dir/Ref/k50.Umap.MultiTrackMappability.hg38.bw",
'--chrom_sizes_path', "$griffin_code_dir/Ref/hg38.standard.chrom.sizes",
'--tmp_dir', $args{tmpdir},
'--griffin_scripts', "$griffin_code_dir/scripts",
'--sites_yaml', $args{sites},
'--position_column position',
'--size_range 90 220 --map_quality 20 --number_of_sites none',
'--CPU', $args{n_cpus}
);
my $uncorrected_bw = join('/', $args{tmpdir}, $args{id}, 'tmp_bigWig', $args{id} . '.uncorrected.bw');
my $corrected_bw = join('/', $args{tmpdir}, $args{id}, 'tmp_bigWig', $args{id} . '.GC_corrected.bw');
my $map_corrected_bw = join('/', $args{tmpdir}, $args{id}, 'tmp_bigWig', $args{id} . '.GC_map_corrected.bw');
my $exclude_encode = join('/', $griffin_code_dir, 'Ref', 'encode_unified_GRCh38_exclusion_list.bed');
my $exclude_centromeres = join('/', $griffin_code_dir, 'Ref', 'hg38_centromeres.bed');
my $exclude_gaps = join('/', $griffin_code_dir, 'Ref', 'hg38_gaps.bed');
my $exclude_patches = join('/', $griffin_code_dir, 'Ref', 'hg38_fix_patches.bed');
my $exclude_alts = join('/', $griffin_code_dir, 'Ref', 'hg38_alternative_haplotypes.bed');
$griffin_command .= "\n\n" . join(' ',
"$griffin_code_dir/scripts/griffin_merge_sites.py",
'--sample_name', $args{id},
'--uncorrected_bw_path', $uncorrected_bw,
'--GC_corrected_bw_path', $corrected_bw,
'--GC_map_corrected_bw_path', $map_corrected_bw,
'--mappability_correction False',
'--mappability_bw', "$griffin_code_dir/Ref/k50.Umap.MultiTrackMappability.hg38.bw",
'--chrom_sizes_path', "$griffin_code_dir/Ref/hg38.standard.chrom.sizes",
'--sites_yaml', $args{sites},
'--griffin_scripts_dir', "$griffin_code_dir/scripts",
'--tmp_dir', $args{tmpdir},
'--results_dir', $args{outdir},
'--position_column position',
'--exclude_paths', $exclude_encode, $exclude_centromeres, $exclude_gaps, $exclude_patches, $exclude_alts,
'--exclude_outliers True',
'--exclude_zero_mappability True',
'--step 15',
'--CNA_normalization False',
'--CPU', $args{n_cpus},
'--individual False',
'--smoothing True'
);
}
$griffin_command .= "\n\n" . join(' ',
"echo 'Griffin nucleosome profiling completed successfully' >",
$args{outdir} . '/nucleosome_profiling.COMPLETE'
);
return($griffin_command);
}
# format command to run End Motif profiling
sub get_end_motif_command {
my %args = (
id => undef,
bedpe => undef,
outdir => undef,
@_
);
my $output_stem = join('/', $args{outdir}, $args{id});
my $linked_bedpe = join('/', $args{outdir}, $args{id} . '.bedpe' );
my $motif_command = "ln -s $args{bedpe} $linked_bedpe";
# format bedpe to bed
$motif_command .= "\n\n# convert bedpe to bed";
$motif_command .= "\necho '>>> Running motif_format_bedpe.R <<<'";
$motif_command .= "\n\n" . join(' ',
"Rscript $fragmentomics_code_dir/end_motif/scripts/motif_format_bedpe.R",
'--id', $args{id},
'--bedpe', $linked_bedpe,
'--outdir', $args{outdir}
);
# get fasta sequences
$motif_command .= "\n\n# extract fasta sequences";
$motif_command .= "\necho '>>> Running bedtools getfasta <<<'";
$motif_command .= "\n\n" . join(' ',
'bedtools getfasta',
'-bedOut -fi', $reference,
'-bed', $output_stem . '_5.bed',
'>', $output_stem . '_fasta_5.bed'
);
$motif_command .= "\n\n" . join(' ',
'bedtools getfasta',
'-bedOut -fi', $reference,
'-bed', $output_stem . '_3.bed',
'>', $output_stem . '_fasta_3.bed'
);
# convert fasta to end motif context frequencies
$motif_command .= "\n\n# run end motif context profiling";
$motif_command .= "\necho '>>> Running motif_get_contexts.R <<<'";
$motif_command .= "\n\n" . join(' ',
"Rscript $fragmentomics_code_dir/end_motif/scripts/motif_get_contexts.R",
"--id", $args{id},
"--fasta_5", $output_stem . '_fasta_5.bed',
"--fasta_3", $output_stem . '_fasta_3.bed',
"--outdir", $args{outdir}
);
# remove intermediate files
$motif_command .= "\n\n# check for completeness and remove intermediates";
$motif_command .= "\n" . join("\n",
'if [[ -f ' . $output_stem . '_motifs.txt ]]; then',
" echo '>>> End Motif Profiling completed successfully! <<<'",
' rm ' . $output_stem . '*.bed*',
'fi'
);
return($motif_command);
}
# format command to run breakpoint profiling
sub get_profile_breakpoints_command {
my %args = (
id => undef,
bedpe => undef,
outdir => undef,
@_
);
my $output_stem = join('/', $args{outdir}, $args{id});
my $linked_bedpe = join('/', $args{outdir}, $args{id} . '.bedpe' );
my $bkpt_command = "ln -s $args{bedpe} $linked_bedpe";
# format bedpe to bed
$bkpt_command .= "\n\n# convert bedpe to bed";
$bkpt_command .= "\necho '>>> Running breakpoint_format_bedpe.R <<<'";
$bkpt_command .= "\n\n" . join(' ',
"Rscript $fragmentomics_code_dir/breakpoint/scripts/breakpoint_format_bedpe.R",
'--id', $args{id},
'--bedpe', $linked_bedpe,
'--outdir', $args{outdir}
);
# get fasta sequences
$bkpt_command .= "\n\n# extract fasta sequences";
$bkpt_command .= "\necho '>>> Running bedtools getfasta <<<'";
$bkpt_command .= "\n\n" . join(' ',
'bedtools getfasta',
'-bedOut -fi', $reference,
'-bed', $output_stem . '_5.bed',
'>', $output_stem . '_fasta_5.bed'
);
$bkpt_command .= "\n\n" . join(' ',
'bedtools getfasta',
'-bedOut -fi', $reference,
'-bed', $output_stem . '_3.bed',
'>', $output_stem . '_fasta_3.bed'
);
# convert fasta to end motif context frequencies
$bkpt_command .= "\n\n# run breakpoint context profiling";
$bkpt_command .= "\necho '>>> Running breakpoint_get_contexts.R <<<'";
$bkpt_command .= "\n\n" . join(' ',
"Rscript $fragmentomics_code_dir/breakpoint/scripts/breakpoint_get_contexts.R",
"--id", $args{id},
"--fasta_5", $output_stem . '_fasta_5.bed',
"--fasta_3", $output_stem . '_fasta_3.bed',
"--outdir", $args{outdir}
);
# remove intermediate files
$bkpt_command .= "\n\n# check for completeness and remove intermediates";
$bkpt_command .= "\n" . join("\n",
'if [[ -f ' . $output_stem . '_ratio.txt ]]; then',
" echo '>>> Breakpoint Profiling completed successfully! <<<'",
' rm ' . $output_stem . '*.bed*',
'fi'
);
return($bkpt_command);
}
# format command to run dinucleotide profiling
sub get_dinucleotide_profiling_command {
my %args = (
id => undef,
bedpe => undef,
outdir => undef,
@_
);
my $output_stem = join('/', $args{outdir}, $args{id});
# format bedpe to bed
my $din_command = "# convert bedpe to bed";
$din_command .= "\necho '>>> Running dinucleotide_format_bedpe.R <<<'";
$din_command .= "\n\n" . join(' ',
"Rscript $fragmentomics_code_dir/dinucleotide/scripts/dinucleotide_format_bedpe.R",
'--id', $args{id},
'--bedpe', $args{bedpe},
'--outdir', $args{outdir}
);
# get fasta sequences
$din_command .= "\n\n# extract fasta sequences";
$din_command .= "\necho '>>> Running bedtools getfasta <<<'";
$din_command .= "\n\n" . join(' ',
'bedtools getfasta',
'-bedOut -fi', $reference,
'-bed', $output_stem . '.bed',
'>', $output_stem . '_fasta.bed'
);
# convert fasta to end motif context frequencies
$din_command .= "\n\n# run dinucleotide context profiling";
$din_command .= "\necho '>>> Running dinucleotide_get_contexts.R <<<'";
$din_command .= "\n\n" . join(' ',
"Rscript $fragmentomics_code_dir/dinucleotide/scripts/dinucleotide_get_contexts.R",
"--id", $args{id},
"--fasta", $output_stem . '_fasta.bed',
"--outdir", $args{outdir}
);
# remove intermediate files
$din_command .= "\n\n# check for completeness and remove intermediates";
$din_command .= "\n" . join("\n",
'if [[ -f ' . $output_stem . '_contexts.txt ]]; then',
" echo '>>> Dinucleotide Profiling completed successfully! <<<'",
#' rm ' . $output_stem . '*.bed*',
'fi'
);
return($din_command);
}
### MAIN ###########################################################################################
sub main {
my %args = (
tool_config => undef,
data_config => undef,
output_directory => undef,
hpc_driver => undef,
dry_run => undef,
no_wait => undef,
@_
);
my $tool_config = $args{tool_config};
my $data_config = $args{data_config};
### PREAMBLE ######################################################################################
unless($args{dry_run}) {
print "Initiating Fragmenomics pipeline...\n";
}
# load tool config
my $tool_data = LoadFile($tool_config);
# organize output and log directories
my $output_directory = $args{output_directory};
$output_directory =~ s/\/$//;
my $log_directory = join('/', $output_directory, 'logs');
unless(-e $log_directory) { make_path($log_directory); }
my $log_file = join('/', $log_directory, 'run_Fragmentomics_pipeline.log');
# create a file to hold job metrics
my (@files, $run_count, $outfile, $touch_exit_status);
unless ($args{dry_run}) {
# initiate a file to hold job metrics
opendir(LOGFILES, $log_directory) or die "Cannot open $log_directory";
@files = grep { /slurm_job_metrics/ } readdir(LOGFILES);
$run_count = scalar(@files) + 1;
closedir(LOGFILES);
$outfile = $log_directory . '/slurm_job_metrics_' . $run_count . '.out';
$touch_exit_status = system("touch $outfile");
if (0 != $touch_exit_status) { Carp::croak("Cannot touch file $outfile"); }
$log_file = join('/', $log_directory, 'run_Fragmentomics_pipeline_' . $run_count . '.log');
}
# start logging
open (my $log, '>', $log_file) or die "Could not open $log_file for writing.";
$log->autoflush;
print $log "---\n";
print $log "Running Fragmentomics pipeline.\n";
print $log "\n Tool config used: $tool_config";
print $log "\n Sample config used: $data_config";
print $log "\n Output directory: $output_directory";
print $log "\n Reference used: $tool_data->{reference}";
print $log "\n---";
# get user-specified tool parameters
my $parameters = $tool_data->{parameters};
# set tools and versions
my $picard = 'picard/' . $tool_data->{picard_version};
my $sambamba = 'sambamba/' . $tool_data->{sambamba_version};
my $samtools = 'samtools/' . $tool_data->{samtools_version};
my $bedtools = 'bedtools/' . $tool_data->{bedtools_version};
my $python3 = 'python3/' . $tool_data->{python3_version};
my $r_version = 'R/' . $tool_data->{r_version};
if (defined($tool_data->{fragmentomics_dir})) {
$fragmentomics_code_dir = $tool_data->{fragmentomics_dir};
} else {
$fragmentomics_code_dir = "$cwd/scripts/fragmentomics";
}
if (defined($tool_data->{griffin_dir})) {
$griffin_code_dir = $tool_data->{griffin_dir};
} else {
$griffin_code_dir = "$cwd/scripts/fragmentomics/griffin";
}
# set reference files
$reference = $tool_data->{reference};
$reference_peaks = defined($tool_data->{nucleosome_peaks}) ? $tool_data->{nucleosome_peaks} : "$fragmentomics_code_dir/nucleosome_peak/ref/hg38";
$delfi_filters = defined($tool_data->{delfi_filters}) ? $tool_data->{delfi_filters} : "$fragmentomics_code_dir/ratio/extdata/filters.hg38.rda";
$delfi_gaps = defined($tool_data->{delfi_gaps}) ? $tool_data->{delfi_gaps} : "$fragmentomics_code_dir/ratio/extdata/gaps.hg38.rda";
$delfi_vntrs = defined($tool_data->{delfi_vntrs}) ? $tool_data->{delfi_vntrs} : "$fragmentomics_code_dir/ratio/extdata/VNTRs.hg38.rda";
$delfi_tiles = defined($tool_data->{delfi_tiles}) ? $tool_data->{delfi_tiles} : "$fragmentomics_code_dir/ratio/extdata/hg38_tiles.bed";
$delfi_healthy = defined($tool_data->{delfi_pon}) ? $tool_data->{delfi_pon} : "$fragmentomics_code_dir/ratio/extdata/healthy.median.hg38.rda";
if (defined($tool_data->{griffin_ref_mappable})) {
$griffin_mappable = $tool_data->{griffin_ref_mappable};
} else {
$griffin_mappable = "$cwd/scripts/fragmentomics/griffin/Ref/repeat_masker.mapable.k50.Umap.hg38.bedGraph.gz";
}
$griffin_mappable_name = basename $griffin_mappable;
$griffin_mappable_name =~ s/\.[^.]+$//;
# use custom list of griffin sites
if (defined($tool_data->{griffin_sites})) {
$griffin_sites = $tool_data->{griffin_sites};
}
# or use default options (will always run TFBS/TCGA/DHS/housekeeping
if (!defined($griffin_sites->{TFBS})) {
$griffin_sites->{TFBS} = "$griffin_code_dir/site_configs/TFBS_sites.yaml";
}
if (!defined($griffin_sites->{TCGA})) {
$griffin_sites->{TCGA} = "$griffin_code_dir/site_configs/TCGA_sites.yaml";
}
if (!defined($griffin_sites->{DHS})) {
$griffin_sites->{DHS} = "$griffin_code_dir/site_configs/DHS_sites.yaml";
}
if (!defined($griffin_sites->{housekeeping})) {
$griffin_sites->{housekeeping} = "$griffin_code_dir/site_configs/housekeeping_sites.yaml";
}
# get optional HPC group
my $hpc_group = defined($tool_data->{hpc_group}) ? "-A $tool_data->{hpc_group}" : undef;
### RUN ###########################################################################################
# first, determine which steps/fragmentomics analyses to run
my %tool_set = (
'downsample' => 'Y', #defined($parameters->{downsample}) ? 'Y' : 'N',
'deduplicate' => 'N', #defined($parameters->{dedup}) ? 'Y' : 'N',
'ratio' => defined($parameters->{ratio}) ? 'Y' : 'N',
'score' => defined($parameters->{score}) ? 'Y' : 'N',
'insertsize' => defined($parameters->{insertsize}) ? 'Y' : 'N',
'griffin' => defined($parameters->{griffin}) ? 'Y' : 'N',,
'nucleosome' => defined($parameters->{nucleosome_position}) ? 'Y' : 'N',
'dinucleotide' => defined($parameters->{dinucleotide}) ? 'Y' : 'N',
'end_motifs' => defined($parameters->{end_motifs}) ? 'Y' : 'N',
'breakpoints' => defined($parameters->{breakpoints}) ? 'Y' : 'N'
);
if ( ('Y' eq $tool_set{'nucleosome'}) ||
('Y' eq $tool_set{'end_motifs'}) || ('Y' eq $tool_set{'breakpoints'}) ) {
$tool_set{'deduplicate'} = 'Y';
}
print $log "\nTools/steps to run:\n";
print $log Dumper \%tool_set;
# get sample data
my $smp_data = LoadFile($data_config);
unless($args{dry_run}) {
print "Processing " . scalar(keys %{$smp_data}) . " patients.\n";
}
# extract downsample parameters
my ($n_reads, $scale_factor);
if (defined($parameters->{downsample}->{n_reads})) {
$n_reads = $parameters->{downsample}->{n_reads};
} elsif (defined($parameters->{downsample}->{scale_factor})) {
$scale_factor = $parameters->{downsample}->{scale_factor};
} else {
$n_reads = 50000000;
print "\nNo scaling parameters defined for downsampling; will default to 50M reads. Alternatively, you can specifiy these (number of reads (n_reads) or scaling factor (scale_factor)) in your tool config.";
}
# initialize objects
my ($run_script, $run_id);
my @all_jobs;
# process each patient in $smp_data
foreach my $patient (sort keys %{$smp_data}) {
print $log "\n---\nInitiating process for PATIENT: $patient";
# find bams
my @tumour_ids = sort keys %{$smp_data->{$patient}->{'tumour'}};
if (scalar(@tumour_ids) == 0) {
print $log "\n>> No tumour BAM provided, skipping patient.\n";
next;
}
# create some directories
my $patient_directory = join('/', $output_directory, $patient);
my $tmp_directory = join('/', $patient_directory, 'TEMP');
unless(-e $patient_directory) { make_path($patient_directory); }
unless(-e $tmp_directory) { make_path($tmp_directory); }
my (@final_outputs, @patient_jobs);
my $cleanup_cmd = " rm -rf $tmp_directory;";
# process each sample provided for this patient
foreach my $tumour (@tumour_ids) {
# initiate some variables
my $tumour_stem = $tumour;
my $tumour_bam = $smp_data->{$patient}->{tumour}->{$tumour};
my ($downsample_run_id, $dedup_run_id, $griffin_id) = '';
# set up directory structure
my $sample_directory = join('/', $patient_directory, $tumour);
my $downsample_directory = join('/', $sample_directory, 'downsample');
my $ratio_directory = join('/', $sample_directory, 'fragment_ratio');
my $score_directory = join('/', $sample_directory, 'fragment_score');
my $insertsize_directory = join('/', $sample_directory, 'insert_size');
my $nucleosome_directory = join('/', $sample_directory, 'nucleosome_peaks');
my $griffin_directory = join('/', $sample_directory, 'griffin');
my $motif_directory = join('/', $sample_directory, 'end_motifs');
my $breakpoint_directory = join('/', $sample_directory, 'breakpoints');
my $di_directory = join('/', $sample_directory, 'dinucleotide');
unless(-e $sample_directory) { make_path($sample_directory); }
if ('Y' eq $tool_set{'downsample'}) {
unless(-e $downsample_directory) { make_path($downsample_directory); }
}
if ('Y' eq $tool_set{'ratio'}) {
unless(-e $ratio_directory) { make_path($ratio_directory); }
}
if ('Y' eq $tool_set{'score'}) {
unless(-e $score_directory) { make_path($score_directory); }
}
if ('Y' eq $tool_set{'insertsize'}) {
unless(-e $insertsize_directory) { make_path($insertsize_directory); }
}
if ('Y' eq $tool_set{'nucleosome'}) {
unless(-e $nucleosome_directory) { make_path($nucleosome_directory); }
}
if ('Y' eq $tool_set{'griffin'}) {
unless(-e $griffin_directory) { make_path($griffin_directory); }
}
if ('Y' eq $tool_set{'end_motifs'}) {
unless(-e $motif_directory) { make_path($motif_directory); }
}
if ('Y' eq $tool_set{'breakpoints'}) {
unless(-e $breakpoint_directory) { make_path($breakpoint_directory); }
}
if ('Y' eq $tool_set{'dinucleotide'}) {
unless(-e $di_directory) { make_path($di_directory); }
}
# run downsample
if ('Y' eq $tool_set{'downsample'}) {
my $downsample_cmd = create_downsample_command(
bam => $smp_data->{$patient}->{tumour}->{$tumour},
id => $tumour,