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psirc_v1.0.pl
executable file
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psirc_v1.0.pl
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#!/usr/bin/perl
use strict; use warnings;
use Getopt::Long qw(GetOptions);
##
#BEGIN { unshift @INC, "/home/hoyu/scripts"; }
#use DataBrowser;
##
my $version = "_v1.0";
my $kallisto_fork = "kallisto_b";
#Do these sam flags indicate they are first or second in pair?
my %in_pair = (
99 => '1',
355 => '1',
147 => '2',
403 => '2',
83 => '1',
339 => '1',
163 => '2',
419 => '2',
69 => '1',
73 => '1',
89 => '1',
101 => '1',
329 => '1',
345 => '1',
77 => '1',
133 => '2',
137 => '2',
153 => '2',
165 => '2',
393 => '2',
409 => '2',
141 => '2',
);
my %transcripts_seen;
my %bsjs_with_equiv_fsj;
my $kallisto_k = 31;
my $kallisto_l = 30;
my $segment_kallisto_l = 15;
my $gen_indices;
my $threads_kallisto = 1;
my $EXPECT_READIDS_PER_BSJ = 2;
#my $DEFAULT_UNIQUE_BY = 10;
my $MBSL = 7;
my $output_dir = ".";
my $final_kallisto_k = 21;
my $straight_to_full_length;
my $equiv_fsj;
my $lib_type = "fr-unstranded";
my $unique_by = 10;
my $linear_ratio_info;
my $max_mappable_bsjs = 300;
my $max_mappable_alt_fsjs = 20;
my $max_alt_fsj_per_isoform = 5;
my $full_length;
GetOptions(
'i|indices' => \$gen_indices,
't|threads=i' => \$threads_kallisto,
'o|output-dir=s' => \$output_dir,
'y|library-type=s' => \$lib_type,
'm|mbsl=i' => \$MBSL,
'r|final-k=i' => \$final_kallisto_k,
'u|unique-by=i' => \$unique_by,
's|straight-to-full-length' => \$straight_to_full_length,
'j|equiv-fsj' => \$equiv_fsj,
'l|linear-ratio-info' => \$linear_ratio_info,
'b|max-mappable-bsjs=i' => \$max_mappable_bsjs,
'a|max-mappable-alt-fsjs=i' => \$max_mappable_alt_fsjs,
'p|max-alt-fsj-per-isoform=i' => \$max_alt_fsj_per_isoform,
'f|full-length' => \$full_length,
) or die_usage();
#segment partition settings
my ($p_1_kmer, $p_1_kmer_bp_low, $p_1_kmer_bp_high, $p_1_kmer_target_length) = ($MBSL, $MBSL, 14, 20);
my ($p_2_kmer, $p_2_kmer_bp_low, $p_2_kmer_bp_high, $p_2_kmer_target_length) = (13, 15, 22, 30);
my ($p_3_kmer, $p_3_kmer_bp_low) = (21, 23);
die "$output_dir does not exist\n" unless -d "$output_dir";
die "Supported library types are fr-unstranded, fr-firststrand, or fr-secondstrand\n"
unless ( ($lib_type eq "fr-unstranded") || ($lib_type eq "fr-firststrand") || ($lib_type eq "fr-secondstrand") );
die "-m/--mbsl must be a whole number between 5 and 10\n" unless ( ($MBSL >= 5) && ($MBSL <= 10) );
die "-r/--final-k must be an odd whole number between 1 and 31\n" unless ( ($final_kallisto_k % 2 == 1)
&& ($final_kallisto_k >= 1) && ($final_kallisto_k <= 31) && ($final_kallisto_k =~ /^\d+\z/) );
die "-f/--full-length cannot be used with -a/--linear-ratio-info. Use the standard run or -s instead\n"
if ($linear_ratio_info && $full_length);
my ($fa, $kallisto, $fq1, $fq2, $provided_dir);
if ($gen_indices) {
die_usage_4() unless (scalar(@ARGV)==2);
die "fasta file names need to end with .fa or .fasta\n" unless ($ARGV[0] =~ /.*(\.fa|\.fasta)$/);
die "$ARGV[0] not found\n" unless (-e $ARGV[0]);
die "$ARGV[1] not found\n" unless (-e $ARGV[1]);
($fa, $kallisto) = @ARGV;
} elsif ($linear_ratio_info) {
die_usage_3() unless (scalar(@ARGV)==5);
die "fasta file names need to end with .fa or .fasta\n" unless ($ARGV[1] =~ /.*(\.fa|\.fasta)$/);
die "$ARGV[0] not found\n" unless (-e $ARGV[0]);
die "$ARGV[1] not found\n" unless (-e $ARGV[1]);
die "$ARGV[2] not found\n" unless (-e $ARGV[2]);
die "$ARGV[3] not found\n" unless (-e $ARGV[3]);
die "$ARGV[4] not found\n" unless (-e $ARGV[4]);
($provided_dir, $fa, $kallisto, $fq1, $fq2) = @ARGV;
} elsif ($straight_to_full_length) {
die_usage_2() unless (scalar(@ARGV)==5);
die "fasta file names need to end with .fa or .fasta\n" unless ($ARGV[1] =~ /.*(\.fa|\.fasta)$/);
die "$ARGV[0] not found\n" unless (-e $ARGV[0]);
die "$ARGV[1] not found\n" unless (-e $ARGV[1]);
die "$ARGV[2] not found\n" unless (-e $ARGV[2]);
die "$ARGV[3] not found\n" unless (-e $ARGV[3]);
die "$ARGV[4] not found\n" unless (-e $ARGV[4]);
($provided_dir, $fa, $kallisto, $fq1, $fq2) = @ARGV;
}
else {
die_usage() unless (scalar(@ARGV)==4);
die "$ARGV[0] not found\n" unless (-e $ARGV[0]);
die "fasta file names need to end with .fa or .fasta\n" unless ($ARGV[0] =~ /.*(\.fa|\.fasta)$/);
die "$ARGV[1] not found\n" unless (-e $ARGV[1]);
die "$ARGV[2] not found\n" unless (-e $ARGV[2]);
die "$ARGV[3] not found\n" unless (-e $ARGV[3]);
($fa, $kallisto, $fq1, $fq2) = @ARGV;
}
if ($straight_to_full_length || $linear_ratio_info) {
$output_dir = $provided_dir;
}
#Remove final slash in output_dir if given
if ($output_dir =~ m/\/$/) {
chop($output_dir);
}
my $mapping_dir = $output_dir . "/mapping";
unless ($gen_indices) {
mkdir $mapping_dir unless (-d $mapping_dir);
}
my $target_potential_backsplice_fasta = $mapping_dir . "/" . "potential_backsplice_transcripts.fa";
my $target_potential_backsplice_fasta_no_copied_seq = $mapping_dir . "/" . "potential_backsplice_transcripts.no_copied_seq.fa";
my $potential_backsplice_reads_1 = $mapping_dir . "/possible_bsj_reads_1.fastq.gz";
my $potential_backsplice_reads_2 = $mapping_dir . "/possible_bsj_reads_2.fastq.gz";
my $low_num_temp_sam = $mapping_dir . "/temp_final_mapping.sam";
my $num_transcripts;
my $low_num_transcripts = 500;
my $num_reads;
my $low_num_reads = 100000;
my $low_num;
my $low_num_sam=0;
my %low_num_read_ids;
my ($fa_file_pre, $full_path_fa_file_pre);
if ($fa =~ m/(.*\/)(.*\.fasta)$/) {
my $path = $1;
my $fa_file = $2;
$fa_file =~ s/\.fasta//;
$fa_file_pre = $fa_file;
$full_path_fa_file_pre = $path . $fa_file_pre;
} elsif ($fa =~ m/(.*\/)(.*\.fa)$/) {
my $path = $1;
my $fa_file = $2;
$fa_file =~ s/\.fa//;
$fa_file_pre = $fa_file;
$full_path_fa_file_pre = $path . $fa_file_pre;
} elsif ($fa =~ m/(.*\.fasta)$/) {
my $fa_file = $1;
$fa_file =~ s/\.fasta//;
$fa_file_pre = $fa_file;
$full_path_fa_file_pre = $fa_file_pre;
} elsif ($fa =~ m/(.*\.fa)$/) {
my $fa_file = $1;
$fa_file =~ s/\.fa//;
$fa_file_pre = $fa_file;
$full_path_fa_file_pre = $fa_file_pre;
}
my $all_possible_bsj_targets_fa = "$full_path_fa_file_pre.all_possible_bsj_targets.fa";
my $FirstandLastExons_entities_fa = "$full_path_fa_file_pre.FirstandLastExons_entities.fa";
my $all_possible_bsj_targets_fa_index = $all_possible_bsj_targets_fa . ".index";
my $FirstandLastExons_entities_fa_index = $FirstandLastExons_entities_fa . ".index";
my $READ_LENGTHS;
if ( ($gen_indices) && (!$linear_ratio_info) && (!$straight_to_full_length)) {
print "Generating all possible bsj and FirstandLastExons entities fa files and their kallisto indices... ";
system("date");
generate_targets_and_indices();
system("date");
die "Finished generating. The indices should be kept in the same file path as the <transcriptome_fa> and their names should not be changed.\n";
} elsif ( ($gen_indices) && ($linear_ratio_info) ) {
die_usage_4();
}
if ( (!$linear_ratio_info) && (!$straight_to_full_length) ) {
$READ_LENGTHS = map_reads_to_targets();
} elsif ( ($straight_to_full_length) && (!$linear_ratio_info) ) {
print "Getting the required variables... ";
system("date");
get_required_variables("s");
print "Ready. ";
system("date");
} elsif ( (!$straight_to_full_length) && ($linear_ratio_info) ) {
print "Getting the required variables... ";
system("date");
get_required_variables("l");
print "Ready. ";
system("date");
}
print "Final mapping... ";
system("date");
###
if ($low_num) {
if ($low_num_sam != 1) {
my $backsplice_transcripts_fa_index = $target_potential_backsplice_fasta . ".index";
system("$kallisto index -k $final_kallisto_k -i $backsplice_transcripts_fa_index $target_potential_backsplice_fasta");
system("$kallisto pseudo -i $backsplice_transcripts_fa_index -o $mapping_dir -t $threads_kallisto --pseudobam $potential_backsplice_reads_1 $potential_backsplice_reads_2 > $low_num_temp_sam");
check_temp_sam($low_num_temp_sam);
}
}
###
my %transcripts_bsjs;
my %outputted_raw_names;
my %raw_name_strand;
final_mapping_and_output_candidate_circRNAs() unless ($straight_to_full_length);
print "Finished mapping. ";
system("date");
print "\n";
#full length
my $full_length_dir = $output_dir . "/full_length";
my $fsj_targets_fa = $full_length_dir . "/$fa_file_pre" . ".within_backspliced.fsj_targets.fa";
my $target_potential_fli_fasta = $full_length_dir . "/" . "potential_full_length_isoforms.fa";
my $invalidated_flis = $full_length_dir . "/". "invalidated_full_length_isoforms.txt";
my %fli_breakpoints;
my %fli_strands;
my ($potential_fsj_reads_1, $potential_fsj_reads_2) = ("$full_length_dir/potential_fsj_reads_1.fastq.gz", "$full_length_dir/potential_fsj_reads_2.fastq.gz");
$low_num_temp_sam = $full_length_dir . "/temp_final_mapping.sam";
$low_num="";
$low_num_sam=0;
if ($full_length || $straight_to_full_length) {
print "Full length isoform inference... ";
system("date");
mkdir $full_length_dir unless (-d $full_length_dir);
generate_alt_fsj_targets();
map_reads_to_targets_fsj();
if ($low_num) {
my $full_length_isoforms_fa_index = $target_potential_fli_fasta . ".index";
system("$kallisto index -k $final_kallisto_k -i $full_length_isoforms_fa_index $target_potential_fli_fasta");
system("$kallisto pseudo -i $full_length_isoforms_fa_index -o $full_length_dir -t $threads_kallisto --pseudobam $potential_fsj_reads_1 $potential_fsj_reads_2 > $low_num_temp_sam");
check_temp_sam($low_num_temp_sam);
}
print "Final mapping... ";
system("date");
final_mapping_and_output_fsjs();
print "Finished full length isoform inference. ";
system("date");
} else {
exit;
}
sub die_usage_4 {
die "
Usage: perl psirc$version.pl -i/--indices <transcriptome_fa> <$kallisto_fork" . "_executable> generates the two kallisto indices
required for psirc input
[outputs to same location as <transcriptome_fa>]
";
}
sub die_usage {
die "
Usage: perl psirc$version.pl [Options] <transcriptome_fa> <$kallisto_fork" . "_executable> <first_in_pair_fastq> <second_in_pair_fastq>
Before using the (above) main script, run the following once to generate the required kallisto indices for <transcriptome_fa>,
Generate indices: perl psirc$version.pl -i/--indices <transcriptome_fa> <$kallisto_fork" . "_executable> generates the two kallisto indices
required for psirc input
[outputs to same location as <transcriptome_fa>]
Options:
-o/--output-dir <string> the output directory [default ./]
-t/--threads <int> number of threads to run kallisto with [default $threads_kallisto]
-u/--unique-by <int> max number of genomic bsj loci a read is allowed to be mapped to [default $unique_by]
-y/--library-type <lib_type> the library type of the paired input reads [default $lib_type]
-m/--mbsl <int> minimum back-splice segment length [default $MBSL]
-r/--final-k <int> the kallisto k-mer size used for final remapping [default $final_kallisto_k]
-j/--equiv-fsj check if the output the candidate circRNA junction also supports an fsj [default FALSE]
-s/--straight-to-full-length straight to full-length isoform inference by providing default run outdir [default FALSE]
-l/--linear-ratio-info use all the original reads in final step, output linear read counts file [default FALSE]
-b/--max-mappable-bsjs maximum number of mappable back-splice junctions for a transcript [default $max_mappable_bsjs]
-a/--max-mappable-alt-fsjs maximum number of mappable alt forward-splice junctions for a transcript [default $max_mappable_alt_fsjs]
-p/--max-alt-fsj-per-isoform maximum alt forward-splice junctions allowed per transcript isoform [default $max_alt_fsj_per_isoform]
-f/--full-length infer full-length circRNA isoforms **necessary for input to psirc-quant** [default FALSE]
<lib_type>:
fr-unstranded
fr-firststrand
fr-secondstrand
";
}
sub die_usage_2 {
die "
Usage: perl psirc$version.pl -s/--straight-to-full-length [Options] <default_run_output_dir> <transcriptome_fa> <$kallisto_fork" . "_executable> <first_in_pair_fastq> <second_in_pair_fastq>
Options:
-t/--threads <int> number of threads to run kallisto with [default $threads_kallisto]
-u/--unique-by <int> max number of genomic bsj loci a read is allowed to be mapped to [default $unique_by]
-y/--library-type <lib_type> the library type of the paired input reads [default $lib_type]
-m/--mbsl <int> minimum back-splice segment length [default $MBSL]
-r/--final-k <int> the kallisto k-mer size used for final remapping [default $final_kallisto_k]
-j/--equiv-fsj check if the output the candidate circRNA junction also supports an fsj [default FALSE]
-l/--linear-ratio-info use all the original reads in final step, output linear read counts file [default FALSE]
-b/--max-mappable-bsjs maximum number of mappable back-splice junctions for a transcript [default $max_mappable_bsjs]
-a/--max-mappable-alt-fsjs maximum number of mappable alt forward-splice junctions for a transcript [default $max_mappable_alt_fsjs]
-p/--max-alt-fsj-per-isoform maximum alt forward-splice junctions allowed per transcript isoform [default $max_alt_fsj_per_isoform]
<lib_type>:
fr-unstranded
fr-firststrand
fr-secondstrand
";
}
sub die_usage_3 {
die "
Usage: perl psirc$version.pl -l/--linear-ratio-info [Options] <default_run_output_dir> <transcriptome_fa> <$kallisto_fork" . "_executable> <first_in_pair_fastq> <second_in_pair_fastq>
Options:
-t/--threads <int> number of threads to run kallisto with [default $threads_kallisto]
-u/--unique-by <int> max number of genomic bsj loci a read is allowed to be mapped to [default $unique_by]
-y/--library-type <lib_type> the library type of the paired input reads [default $lib_type]
-m/--mbsl <int> minimum back-splice segment length [default $MBSL]
-r/--final-k <int> the kallisto k-mer size used for final remapping [default $final_kallisto_k]
-j/--equiv-fsj check if the output the candidate circRNA junction also supports an fsj [default FALSE]
-b/--max-mappable-bsjs maximum number of mappable back-splice junctions for a transcript [default $max_mappable_bsjs]
-a/--max-mappable-alt-fsjs maximum number of mappable alt forward-splice junctions for a transcript [default $max_mappable_alt_fsjs]
-p/--max-alt-fsj-per-isoform maximum alt forward-splice junctions allowed per transcript isoform [default $max_alt_fsj_per_isoform]
<lib_type>:
fr-unstranded
fr-firststrand
fr-secondstrand
";
}
sub get_required_variables {
my $type=shift;
#get the hash for existing transcripts first
open IN, "<$fa" or die "can't open $fa\n";
while (<IN>) {
chomp;
if ($_ =~ m/^.*:\d+-\d+_(.*)_Exon_Lengths_.*_Offsets_.*_/) {
my $pure_name = $1;
my ($transcript_name) = $_ =~ m/^>(.*)/;
if ( (exists $transcripts_seen{$pure_name}) && ($transcripts_seen{$pure_name} ne $transcript_name) ) {
$transcripts_seen{$transcript_name} = $transcript_name;
$pure_name = $transcript_name;
}
else {
$transcripts_seen{$pure_name} = $transcript_name;
}
}
}
close IN;
my $outputted_raw_names_list = $mapping_dir . "/outputted_raw_names.lst";
if ($type eq "s") {
open IN, "<$outputted_raw_names_list" or die "can't open $outputted_raw_names_list\n";
while (<IN>) {
chomp;
my @line = split("\t", $_);
my ($raw_name, $num_reads) = ($line[0], $line[1]);
$outputted_raw_names{$raw_name} = $num_reads;
my ($pure_name, $bsj);
if ($raw_name =~ m/^(.*)_(E\d+B\d+)$/) {
$pure_name = $1;
$bsj = $2;
}
my ($e, $b);
if ($bsj =~ m/E(\d+)B(\d+)/) {
$e = $1;
$b = $2;
}
my $transcript_name = $transcripts_seen{$pure_name};
my $strand;
if ($transcript_name =~ m/^.*:\d+-\d+_.*_Exon_Lengths_.*_Offsets_.*_(\+|-)/) {
$strand = $1;
}
$raw_name_strand{$raw_name} = $strand;
if ( ($e - $b) == 0 ) {
next;
}
$transcripts_bsjs{$transcript_name}{$bsj}++;
}
close IN;
}
my %read_lengths;
incr_read_lengths(\%read_lengths, "$output_dir/mapping/possible_bsj_reads_1.fastq.gz");
incr_read_lengths(\%read_lengths, "$output_dir/mapping/possible_bsj_reads_2.fastq.gz");
for my $read_length (sort { $read_lengths{$b} <=> $read_lengths{$a} } keys %read_lengths) {
$READ_LENGTHS = $read_length;
last;
}
print "Most common read length: $READ_LENGTHS\n";
}
sub incr_read_lengths {
my ($read_lengths, $bsj_reads_fq) = @_;
open IN, "gunzip -c $bsj_reads_fq |" or die "can't open $bsj_reads_fq\n";
my $line=1;
while (<IN>) {
if ($line == 2) {
chomp($_);
$read_lengths->{length($_)}++;
}
if ($line==4) {
$line=0;
}
$line++;
}
close IN;
}
sub populate_and_extract_final_step_exon_ends_fasta {
my ($backsplice_candidate_name_breakpoint_bp, $type) = @_;
my %exon_ends_fasta_p1;
my %exon_ends_fasta_p2;
my %exon_ends_fasta_p3;
if ($type eq "bsj") {
open IN, "<$target_potential_backsplice_fasta" or die "can't open $target_potential_backsplice_fasta\n";
} elsif ($type eq "fsj") {
open IN, "<$target_potential_fli_fasta" or die "can't open $target_potential_fli_fasta\n";
}
while (<IN>) {
chomp;
my $backsplice_candidate_name;
if ($_ =~ /^>(.*)/) {
next unless (exists $backsplice_candidate_name_breakpoint_bp->{$1});
$backsplice_candidate_name = $1;
my $backsplice_candidate_seq = <IN>;
my @breakpoint_bps;
my @name_and_begs;
my @name_and_ends;
if ($type eq "bsj") {
push @breakpoint_bps, $backsplice_candidate_name_breakpoint_bp->{$backsplice_candidate_name};
push @name_and_begs, $backsplice_candidate_name . "&beg";
push @name_and_ends, $backsplice_candidate_name . "&end";
} else {
for my $fsj (keys %{$backsplice_candidate_name_breakpoint_bp->{$backsplice_candidate_name}}) {
push @breakpoint_bps, $backsplice_candidate_name_breakpoint_bp->{$backsplice_candidate_name}->{$fsj};
my $name_and_beg = $backsplice_candidate_name . "_" . $fsj . "&beg";
my $name_and_end = $backsplice_candidate_name . "_" . $fsj . "&end";;
push @name_and_begs, $name_and_beg;
push @name_and_ends, $name_and_end;
}
}
for (my $i=0; $i < @breakpoint_bps; $i++) {
my ($max_end_seq_offset, $max_end_seq_length, $max_beg_seq_length);
if ($type eq "bsj") {
$max_end_seq_offset = 0;
$max_end_seq_length = $breakpoint_bps[$i];
$max_beg_seq_length = $breakpoint_bps[$i];
} elsif ( ($breakpoint_bps[$i] - ($READ_LENGTHS-1)) < 0 ) {
$max_end_seq_offset = 0;
$max_end_seq_length = $breakpoint_bps[$i];
$max_beg_seq_length = $breakpoint_bps[$i];
} else {
$max_end_seq_offset = $breakpoint_bps[$i] - ($READ_LENGTHS-1);
$max_end_seq_length = $READ_LENGTHS-1;
$max_beg_seq_length = $READ_LENGTHS-1;
}
my $max_end_seq = substr($backsplice_candidate_seq, $max_end_seq_offset, $max_end_seq_length);
my $max_beg_seq = substr($backsplice_candidate_seq, $breakpoint_bps[$i], $max_beg_seq_length);
my ($p_1_end_seq, $p_1_beg_seq);
if ($max_beg_seq_length < $p_1_kmer_target_length) {
$p_1_end_seq = $max_end_seq;
$p_1_beg_seq = $max_beg_seq;
} else {
$p_1_end_seq = substr($max_end_seq, -($p_1_kmer_target_length));
$p_1_beg_seq = substr($max_beg_seq, 0, $p_1_kmer_target_length);
}
my ($p_2_end_seq, $p_2_beg_seq);
if ($max_beg_seq_length < $p_2_kmer_target_length) {
$p_2_end_seq = $max_end_seq;
$p_2_beg_seq = $max_beg_seq
} else {
$p_2_end_seq = substr($max_end_seq, -($p_2_kmer_target_length));
$p_2_beg_seq = substr($max_beg_seq, 0, $p_2_kmer_target_length);
}
$exon_ends_fasta_p3{$max_end_seq}{$name_and_ends[$i]}++;
$exon_ends_fasta_p3{$max_beg_seq}{$name_and_begs[$i]}++;
$exon_ends_fasta_p2{$p_2_end_seq}{$name_and_ends[$i]}++;
$exon_ends_fasta_p2{$p_2_beg_seq}{$name_and_begs[$i]}++;
$exon_ends_fasta_p1{$p_1_end_seq}{$name_and_ends[$i]}++;
$exon_ends_fasta_p1{$p_1_beg_seq}{$name_and_begs[$i]}++;
}
}
}
close IN;
my $output_exon_ends_p1 = extract_final_step_exon_ends_fasta(\%exon_ends_fasta_p1, "1", $type);
my $output_exon_ends_p2 = extract_final_step_exon_ends_fasta(\%exon_ends_fasta_p2, "2", $type);
my $output_exon_ends_p3 = extract_final_step_exon_ends_fasta(\%exon_ends_fasta_p3, "3", $type);
return ($output_exon_ends_p1, $output_exon_ends_p2, $output_exon_ends_p3);
}
sub extract_final_step_exon_ends_fasta {
my ($exon_ends_fasta, $p, $type) = @_;
my $output_file;
if ($type eq "bsj") {
$output_file = $mapping_dir . "/potential_backsplice_transcripts.final_step.junctions.p$p.fa";
} elsif ($type eq "fsj") {
$output_file = $full_length_dir . "/fsj.final_step.junctions.p$p.fa";
}
open OUT, ">$output_file" or die "can't open $output_file\n";
for my $beg_or_end_seq (keys %{$exon_ends_fasta}) {
my @equiv_headers = keys (%{$exon_ends_fasta->{$beg_or_end_seq}});
my $header = ">" . join("=", @equiv_headers);
print OUT "$header\n$beg_or_end_seq\n";
}
close OUT;
return $output_file;
}
sub print_segment_fastq_seq {
my ($segment_fastq_seq, $type) = @_;
my $final_remapping;
if ($type eq "bsj") {
$final_remapping = $mapping_dir;
} elsif ($type eq "fsj") {
$final_remapping = $full_length_dir;
}
my $prefix = "$final_remapping/segments_final_step_junctions_";
my $output_p1_seg = $prefix . $p_1_kmer_bp_low . "to" . $p_1_kmer_bp_high . ".fastq.gz";
my $output_p2_seg = $prefix . $p_2_kmer_bp_low . "to" . $p_2_kmer_bp_high . ".fastq.gz";
my $output_p3_seg = $prefix . $p_3_kmer_bp_low . "orabove.fastq.gz";
open P1, "| gzip -c > $output_p1_seg" or die "can't open $output_p1_seg\n";
open P2, "| gzip -c > $output_p2_seg" or die "can't open $output_p2_seg\n";
open P3, "| gzip -c > $output_p3_seg" or die "can't open $output_p3_seg\n";
for my $line_2_4 (keys %{$segment_fastq_seq}) {
for my $read_ids (keys %{$segment_fastq_seq->{$line_2_4}}) {
my @final_segment_info_ar;
for my $segment_info (keys %{$segment_fastq_seq->{$line_2_4}->{$read_ids}}) {
push @final_segment_info_ar, $segment_info;
}
my $final_segment_info = join("=", @final_segment_info_ar);
my $line_1 = $read_ids . "=>" . $final_segment_info;
my @line_2_4_info = split("\n", $line_2_4);
my $seg_length = length($line_2_4_info[0]);
if ($seg_length < $p_2_kmer_bp_low) {
print P1 "@" . $line_1 . "\n" . $line_2_4 . "\n";
} elsif ($seg_length < $p_3_kmer_bp_low) {
print P2 "@" . $line_1 . "\n" . $line_2_4 . "\n";
} else {
print P3 "@" . $line_1 . "\n" . $line_2_4 . "\n";
}
}
}
close P1;
close P2;
close P3;
return ($output_p1_seg, $output_p2_seg, $output_p3_seg);
}
sub map_and_examine_final_segments {
my ($output_exon_ends_fasta, $segments_fastq_file, $segment_kallisto_k, $checked_junctions, $type) = @_;
my $final_remapping;
if ($type eq "bsj") {
$final_remapping = $mapping_dir;
} elsif ($type eq "fsj") {
$final_remapping = $full_length_dir;
}
my $output_exon_ends_fa_index = $output_exon_ends_fasta . ".index";
system("$kallisto index -i $output_exon_ends_fa_index -k $segment_kallisto_k $output_exon_ends_fasta");
open IN, "$kallisto pseudo -i $output_exon_ends_fa_index -o $final_remapping -t $threads_kallisto --single -l $segment_kallisto_l -s 20 --pseudobam $segments_fastq_file |" or die "can't run kallisto pseudo segments\n";
my $first_line = <IN>;
my %target_lengths;
while (<IN>) {
next if ($_ =~ /^\@PG/);
if ($_ =~ /^@/) {
my @line = split("\t", $_);
chomp(@line);
my ($length, $target);
if ($line[2] =~ /LN:(\d+)/) {
$length = $1;
}
if ($line[1] =~ /SN:(.*)/) {
$target = $1;
}
$target_lengths{$target} = $length;
next;
}
my @line = split(/\s+/, $_);
my $sam_flag = $line[1];
next if ($sam_flag == 4);
my ($segments, $exon_beg_or_ends, $sam_offset, $sam_cigar) = ($line[0], $line[2], $line[3], $line[5]);
my ($cigar_M) = $sam_cigar =~ /(\d+)M/;
my @segments_info_ar = split("=>", $segments);
my $read_id = $segments_info_ar[0];
my $mapped_segments = $segments_info_ar[1];
my @mapped_segments_ar = split("=", $mapped_segments);
my @exon_beg_or_ends_ar = split("=", $exon_beg_or_ends);
my @read_id_info = split("&", $read_id);
my ($in_pair, $raw_read_id) = ($read_id_info[0], $read_id_info[1]);
for my $mapped_segment (@mapped_segments_ar) {
for my $exon_beg_or_end (@exon_beg_or_ends_ar) {
if ($mapped_segment eq $exon_beg_or_end) {
my @indiv_segment_info_ar = split("&", $mapped_segment);
my ($backsplice_candidate_name, $segment_beg_or_end) = ($indiv_segment_info_ar[0], $indiv_segment_info_ar[1]);
##filter out false positives
if ( ($segment_beg_or_end eq "beg") && ($sam_offset != 1) ) {
next;
}
my $target_length = $target_lengths{$exon_beg_or_ends};
if ($segment_beg_or_end eq "end") {
if ( ($sam_offset + $cigar_M) != ($target_length+1) ) {
next;
}
}
my $in_pair_beg_or_end = $in_pair . $segment_beg_or_end;
if ($type eq "bsj") {
$checked_junctions->{$raw_read_id}->{$backsplice_candidate_name}->{$in_pair_beg_or_end}++;
} else {
$checked_junctions->{$backsplice_candidate_name}->{$raw_read_id}->{$in_pair_beg_or_end}++;
}
}
}
}
}
close IN;
}
sub final_mapping_and_output_candidate_circRNAs {
my $backsplice_transcripts_fa_index = $target_potential_backsplice_fasta . ".index";
system("$kallisto index -k $final_kallisto_k -i $backsplice_transcripts_fa_index $target_potential_backsplice_fasta") unless ($linear_ratio_info);
my $final_remapping;
if ($linear_ratio_info) {
mkdir "$output_dir/linear_ratio_out" unless (-d "$output_dir/linear_ratio_out");
mkdir "$output_dir/linear_ratio_out/mapping" unless (-d "$output_dir/linear_ratio_out/mapping");
$output_dir = "$output_dir/linear_ratio_out";
$final_remapping = "$output_dir/mapping";
} else {
$final_remapping = $mapping_dir;
}
my $proper_pair_out = $final_remapping . "/final_reads_mapped_proper_pair.sam";
open OUT_P, ">$proper_pair_out" or die "can't open $proper_pair_out\n";
######
my $sam_headers = $final_remapping . "/sam_headers";
open OUT_SH, ">$sam_headers" or die "can't open $sam_headers\n";
######
if($linear_ratio_info) {
open IN, "$kallisto pseudo -i $backsplice_transcripts_fa_index -o $final_remapping -t $threads_kallisto --pseudobam $fq1 $fq2 |" or die "can't run kallisto pseudo\n";
} else {
if ( ($low_num) && ($low_num_sam == 1)) {
open IN, "<$low_num_temp_sam" or die "can't open $low_num_temp_sam\n";
} elsif ($low_num) {
open IN, "$kallisto pseudo -i $backsplice_transcripts_fa_index -o $final_remapping -t 1 --pseudobam $potential_backsplice_reads_1 $potential_backsplice_reads_2 |" or die "can't run kallisto pseudo\n";
} else {
open IN, "$kallisto pseudo -i $backsplice_transcripts_fa_index -o $final_remapping -t $threads_kallisto --pseudobam $potential_backsplice_reads_1 $potential_backsplice_reads_2 |" or die "can't run kallisto pseudo\n";
}
}
my %segment_fastq_seq;
my %loci_reads;
my %loci_tx_exons_names;
my %name_reads;
my %raw_to_output;
my %raw_to_loci;
my %potential_olf_reads;
#used for extracting final step exon_ends_fasta
my %backsplice_candidate_name_breakpoint_bp;
my ($first_strand_expectation, $second_strand_expectation);
if ($lib_type eq "fr-firststrand") {
$first_strand_expectation = "-";
$second_strand_expectation = "+";
} elsif ($lib_type eq "fr-secondstrand") {
$first_strand_expectation = "+";
$second_strand_expectation = "-";
}
my %linear_name_reads;
my %linear_loci_reads;
while (<IN>) {
if ($_ =~ m/^@/) {
#################
#################
print OUT_SH $_;
#################
##################
next;
}
my @line = split(/\s+/,$_);
my $sam_flag = $line[1];
if ($line[2] eq "*") {
next;
}
if ( ($sam_flag == 99) || ($sam_flag == 355) || ($sam_flag == 147) || ($sam_flag == 403) || ($sam_flag == 83) || ($sam_flag == 339) || ($sam_flag == 163) || ($sam_flag == 419) ) {
my ($read_id, $backsplice_candidate_name, $sam_start, $sam_cigar, $mate_sam_start) = ($line[0], $line[2], $line[3], $line[5], $line[7]);
my ($pure_name, $e1, $e2);
chomp($backsplice_candidate_name);
if ($backsplice_candidate_name =~ m/^(.*)_E(\d+)B(\d+)$/) {
$pure_name = $1;
$e1 = $2;
$e2 = $3;
}
my ($cigar_M) = $sam_cigar =~ m/(\d+)M/;
my $transcript_name = $transcripts_seen{$pure_name};
my ( $chr, $start, $E_lengths, $E_offs, $strand);
if ($transcript_name =~ m/^(.*):(\d+)-\d+_.*_Exon_Lengths_(.*)_Offsets_(.*)_(\+|-)/) {
$chr = $1;
$start = $2;
$E_lengths = $3;
$E_offs = $4;
$strand = $5;
}
#filter out reads mapped to the unexpected strand if strand specific mode turned on
unless ($lib_type eq "fr-unstranded") {
if ( ($sam_flag == 99) || ($sam_flag == 355) || ($sam_flag == 147) || ($sam_flag == 403) ) {
unless ($strand eq $first_strand_expectation) {
next;
}
} elsif ( ($sam_flag == 83) || ($sam_flag == 339) || ($sam_flag == 163) || ($sam_flag == 419) ) {
unless ($strand eq $second_strand_expectation) {
next;
}
}
}
my @E_lengths_ar = split(",",$E_lengths);
#determine breakpoint position
my $breakpoint_bp;
if ($E_lengths_ar[$e1] < $READ_LENGTHS) {
$breakpoint_bp = $E_lengths_ar[$e1];
} else {
$breakpoint_bp = $READ_LENGTHS-1;
}
#determine linear plus minus region length
my $plus_minus_region_bp;
if ($linear_ratio_info) {
$plus_minus_region_bp = int ( ($breakpoint_bp / 2) + 0.5);
}
#test whether read crossed breakpoint
if ( (($breakpoint_bp - $sam_start) >= ($MBSL - 1)) && (($sam_start + $cigar_M - $breakpoint_bp) >= ($MBSL - 1)) ) {
my @E_offs_ar = split(",",$E_offs);
my $purer_name;
if ($pure_name =~ m/\d+-\d+_/) {
#($purer_name) = $pure_name =~ m/chr.*:\d+-\d+_(.*;.*)_Exon_Lengths.*/;
($purer_name) = $pure_name =~ m/^.*:\d+-\d+_(.*)_Exon_Lengths.*/;
} else {
$purer_name = $pure_name;
}
my $junction;
if ($strand eq "+") {
$junction = $purer_name . "_E" . ($e1+1) . "B" . ($e2+1);
} elsif ($strand eq "-") {
my $tot_exon_num = scalar(@E_lengths_ar);
$junction = $purer_name . "_E" . ($tot_exon_num-$e2) . "B" . ($tot_exon_num-$e1);
}
my $genomic_start = $start + $E_offs_ar[$e2];
my $genomic_end = $start + $E_offs_ar[$e1] + $E_lengths_ar[$e1];
my $loci = "$chr\t$genomic_start\t$genomic_end";
$name_reads{$backsplice_candidate_name}{$read_id}++;
if (!exists $raw_to_output{$backsplice_candidate_name}) {
$raw_to_output{$backsplice_candidate_name} = $junction;
}
if (!exists $raw_to_loci{$backsplice_candidate_name}) {
$raw_to_loci{$backsplice_candidate_name} = $loci;
}
$loci_reads{$loci}{$read_id}{junc}++;
$loci_tx_exons_names{$loci}{$junction}++;
##extract and hash the beg/end segments
my ($read_seq, $read_qual) = ($line[9], $line[10]);
my $end_seg_length = $breakpoint_bp - $sam_start + 1;
my $beg_seg_length = length($read_seq) - $end_seg_length;
my ($end_seg_seq, $end_seg_qual, $beg_seg_seq, $beg_seg_qual);
#if direction of mapped read is "left" (rev compl)
if (($sam_flag == 83) || ($sam_flag == 147) || ($sam_flag == 339) || ($sam_flag == 403) ) {
$read_seq = rev_compl($read_seq);
$read_qual = rev_compl($read_qual);
$beg_seg_seq = substr($read_seq, 0, $beg_seg_length);
$beg_seg_qual = substr($read_qual, 0, $beg_seg_length);
$end_seg_seq = substr($read_seq, $beg_seg_length, $end_seg_length);
$end_seg_qual = substr($read_qual, $beg_seg_length, $end_seg_length);
} else {
$end_seg_seq = substr($read_seq, 0, $end_seg_length);
$end_seg_qual = substr($read_qual, 0, $end_seg_length);
$beg_seg_seq = substr($read_seq, $end_seg_length, $beg_seg_length);
$beg_seg_qual = substr($read_qual, $end_seg_length, $beg_seg_length);
}
my $beg_line_2_4 = "$beg_seg_seq\n+\n$beg_seg_qual";
my $end_line_2_4 = "$end_seg_seq\n+\n$end_seg_qual";
my $in_pair_read_id = $in_pair{$sam_flag} . "&" . $read_id;
my $backsplice_candidate_name_beg = $backsplice_candidate_name . "&beg";
my $backsplice_candidate_name_end = $backsplice_candidate_name . "&end";
$segment_fastq_seq{$beg_line_2_4}{$in_pair_read_id}{$backsplice_candidate_name_beg}++;
$segment_fastq_seq{$end_line_2_4}{$in_pair_read_id}{$backsplice_candidate_name_end}++;
$backsplice_candidate_name_breakpoint_bp{$backsplice_candidate_name} = $breakpoint_bp;
#else if olf
} elsif ( (scalar(@E_lengths_ar) == 1) || (($e1 == (scalar(@E_lengths_ar)-1)) && ($e2 == 0)) ){
my @E_offs_ar = split(",",$E_offs);
#if the read_id for this loci is already determined as junction, skip it
my $genomic_start = $start + $E_offs_ar[$e2];
my $genomic_end = $start + $E_offs_ar[$e1] + $E_lengths_ar[$e1];
my $loci = "$chr\t$genomic_start\t$genomic_end";
if (exists $loci_reads{$loci}{$read_id}) {
next;
}
my $direction;
if (($sam_flag == 83) || ($sam_flag == 147) || ($sam_flag == 339) || ($sam_flag == 403)) {
$direction = "left";
} else {
$direction = "right";
}
if ( ($direction eq "left") || (($linear_ratio_info) && ($direction eq "right")) ) {
#check if read spans the first exon (crossed by BOUNDARY_THRESH) and does not exceed the junction breakpoint
my $first_exon_end = $breakpoint_bp + $E_lengths_ar[0];
if ( (($first_exon_end - $sam_start) >= ($MBSL - 1)) && (($sam_start - $breakpoint_bp) >= 1) ) {
my $incr_string = $in_pair{$sam_flag} . "left";
$potential_olf_reads{$backsplice_candidate_name}{$read_id}{$incr_string}++ unless ($direction eq "right");
if ($linear_ratio_info) {
$linear_loci_reads{$loci}{$read_id}{linF}++;
$linear_name_reads{$backsplice_candidate_name}{$read_id}++;
}
}
}
if ( ($direction eq "right") || (($linear_ratio_info) && ($direction eq "left")) ) {
#dir is right; check if read spans the last exon (crosed by BOUNDARY_THRESH) OR mapped to annealed exon (cannot cross the breapoint)
if (scalar(@E_lengths_ar) == 1) {
if ($sam_start < ($breakpoint_bp - $MBSL)) {
my $incr_string = $in_pair{$sam_flag} . "right";
$potential_olf_reads{$backsplice_candidate_name}{$read_id}{$incr_string}++ unless ($direction eq "left");
if ($linear_ratio_info) {
$linear_loci_reads{$loci}{$read_id}{linF}++;
$linear_name_reads{$backsplice_candidate_name}{$read_id}++;
}
}
elsif ( ($sam_start > $breakpoint_bp) && (($sam_start - $mate_sam_start) >= $cigar_M) ) {
my $incr_string = $in_pair{$sam_flag} . "right";
$potential_olf_reads{$backsplice_candidate_name}{$read_id}{$incr_string}++ unless ($direction eq "left");
if ($linear_ratio_info) {
$linear_loci_reads{$loci}{$read_id}{linF}++;
$linear_name_reads{$backsplice_candidate_name}{$read_id}++;
}
}
} elsif (scalar(@E_lengths_ar) > 1) {
my $second_to_last_exon_end = $breakpoint_bp;
for(my $i=0; $i < scalar(@E_lengths_ar) - 1; $i++) {
$second_to_last_exon_end += $E_lengths_ar[$i];
}
my $sam_end = $sam_start + $cigar_M - 1;
if ($sam_start < ($breakpoint_bp - $MBSL)) {
my $incr_string = $in_pair{$sam_flag} . "right";
$potential_olf_reads{$backsplice_candidate_name}{$read_id}{$incr_string}++ unless ($direction eq "left");
if ($linear_ratio_info) {
$linear_loci_reads{$loci}{$read_id}{linF}++;
$linear_name_reads{$backsplice_candidate_name}{$read_id}++;
}
}
elsif ( (($sam_end - $second_to_last_exon_end) >= $MBSL) && ($sam_start > $breakpoint_bp) && (($sam_start - $mate_sam_start) >= $cigar_M) ) {
my $incr_string = $in_pair{$sam_flag} . "right";
$potential_olf_reads{$backsplice_candidate_name}{$read_id}{$incr_string}++ unless ($direction eq "left");
if ($linear_ratio_info) {
$linear_loci_reads{$loci}{$read_id}{linF}++;
$linear_name_reads{$backsplice_candidate_name}{$read_id}++;
}
}
}
}
} elsif ($linear_ratio_info) {
#test for reads falling in plus_minus_region
if ( ( (($sam_start + $cigar_M) >= $plus_minus_region_bp) && (($sam_start + $cigar_M) <= $breakpoint_bp) ) ||
( ($sam_start >= $breakpoint_bp) && ($sam_start <= ($breakpoint_bp + $plus_minus_region_bp)) )
) {
my @E_offs_ar = split(",",$E_offs);
#if the read_id for this loci is already determined as junction, skip it
my $genomic_start = $start + $E_offs_ar[$e2];
my $genomic_end = $start + $E_offs_ar[$e1] + $E_lengths_ar[$e1];
my $loci = "$chr\t$genomic_start\t$genomic_end";