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CircSimu.pl
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CircSimu.pl
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use 5.012;
use Getopt::Long;
my ($fq1, $fq2, $out, $gtf, $coverage, $coverage2, $rand_mode, $rand_mode2, $read_length, $seq_err, $insert_length, $insert_length2, $perc_minor, $sigma, $sigma2, $ref_dir, $help, $if_chr, $exon_skipping, $psi, $circ_read);
Getopt::Long::GetOptions(
#'1=s' => \$fq1,
#'2=s' => \$fq2,
'O=s' => \$out,
'G=s' => \$gtf,
'C=i' => \$coverage,
'LC=i' => \$coverage2,
'R=i' => \$rand_mode,
'LR=i' => \$rand_mode2,
'L=i' => \$read_length,
'E=i' => \$seq_err,
#'I=i' => \$insert_length,
'D=s' => \$ref_dir,
'CHR1=i' => \$if_chr,
'M=i' => \$insert_length,
'M2=i' => \$insert_length2,
'PM=i' => \$perc_minor,
'S=i' => \$sigma,
'S2=i' => \$sigma2,
'SE=i' => \$exon_skipping,
'PSI=i' => \$psi,
'H!' => \$help
);
srand(5);
my @die_reason;
my $if_die;
#my ($fq1, $fq2, $gtf) = (">>./simulate_80_10X_80bp_200_1.fq",">>./simulate_80_10X_80bp_200_2.fq",">>./gtf_80_10X_80bp_200.out");
#my $coverage = 10;
#my $coverage2 = 100;
#my $rand_mode = 1;
#my $rand_mode2 = 2;
#my $read_length = 80;
my $cRNA_size = 0;
#my $if_PE = 2;
#my $seq_err = 1;
#my $insert_length = 200;
#my $ref_dir = "/panfs/home/zhao/gaoyuan/bwaphage/hg19/";
if(defined($help)){
print "This is CIRI_AS_simulator, a simulation tool for circRNAs. Welcome!\n\n";
print "Written by Yuan Gao. Any questions please mail to gaoyuan06\@mails.ucas.ac.cn.\n\n";
print "Arguments (all required):\n";
#print "\t-1\t\toutput simulated PE reads file 1 name\n";
#print "\t-2\t\toutput simulated PE reads file 2 name\n";
print "\t-O\t\tprefix of output files\n";
print "\t-G\t\tinput gtf formatted annotation file name\n";
print "\t-C\t\tset coverage or max coverage (when choosing -R 2) for circRNAs\n";
print "\t-LC\t\tset coverage or max coverage (when choosing -LR 2) for linear transcripts\n";
print "\t-R\t\tset random mode for circRNAs: 1 for constant coverage; 2 for random coverage\n";
print "\t-LR\t\tset random mode for linear transcripts: 1 for constant coverage; 2 for random coverage\n";
print "\t-L\t\tread length(/bp) of simulated reads (e.g. 100)\n";
print "\t-E\t\tpercentage of sequencing error (e.g. 2)\n";
#print "\t-I\t\tinsertion length (should be larger than read length) (e.g. 350)\n";
print "\t-D\t\tdirectory of reference sequence(s) (please make sure all references referred in gtf file are included in the directory)\n";
print "\t-CHR1\t\tif only choose chr1 to simulate sequencing reads: 1 for yes; 0 for no\n";
print "\t-M\t\taverage(mu/bp) of insert length (major normal distribution) (e.g. 320)\n";
print "\t-M2\t\taverage(mu/bp) of insert length (minor normal distribution) (e.g. 550)\n";
print "\t-PM\t\tpercentage of minor normal distribution in total distribution (e.g. 10; 0 for no minor distribution)\n";
print "\t-S\t\tstandard deviation(sigma/bp) of insert length (e.g. 70)\n";
print "\t-S2\t\tstandard deviation(sigma/bp) of insert length (e.g. 70)\n";
print "\t-SE\t\twhether simulate exon skipping: 1 for yes; 0 for no\n";
print "\t-PSI\t\tpercentage of splice in for skipping exon(-SE should be 1)\n";
print "\t-H\t\tshow help information\n";
$if_die = 1;
}elsif( !defined($out) or !defined($gtf) or !defined($coverage) or !defined($coverage2) or !defined($rand_mode) or !defined($rand_mode2) or !defined($read_length) or !defined($seq_err) or !defined($insert_length) or !defined($ref_dir) or !defined($if_chr) or !defined($sigma) or !defined($exon_skipping) or !defined($psi) ){
$if_die = 2;
#push @die_reason, "Please input complete arguments.\n";
}#elsif($insert_length <= $read_length){
# $if_die = 1;
# print "Insertion length should be larger than read length.\n";
#}
#die if $if_die == 1;
if($sigma <= 0){
push @die_reason, "standard deviation of insert_length should be larger than 0!\n";
}
if($insert_length < $read_length){
push @die_reason, "average of insert_length should be larger than read_length!\n";
}
if($read_length < 40){
push @die_reason, "read_length should be larger than 40!\n";
}
if($sigma >= $insert_length){
push @die_reason, "standard deviation of insert_length should be smaller than average of insert_length!\n";
}
if($psi>=100){
push @die_reason, "percentage of splice in for skipping exon should be smaller than 100!\n";
}elsif($psi<=0 and $exon_skipping == 1){
push @die_reason, "percentage of splice in for skipping exon should be larger than 0!\n";
}
if($if_die == 1){
die "\n";
}elsif($if_die == 2){
die "Please input complete arguments.\n";
}elsif (@die_reason > 0){
print @die_reason;
die;
}
$fq1 = $out."_1.fq";
$fq2 = $out."_2.fq";
$out = $out.".out";
$circ_read = $out."_circ_read.txt";
open FQ1, ">", $fq1 or die "cannot write to $fq1:$!";
open FQ2, ">", $fq2 or die;
open CIRCREAD, ">", $circ_read or die;
print CIRCREAD "circRNA_id\tread_id/bsj_read_id\n";
$insert_length -= $read_length;
my $pi = 3.14159265359;
#$out = ">>./".$out;
my %chr_gene_trsc_exon;
my %chr_gene_trsc_all;
my %chr_gene_trsc_intron;
my %chr_inter;
my %ss_exon_id;
my $ss_element_id;
my $pre_gene = '';
my @gene_anno;
my @chr;
my $seqID;
my $sim_total;
$ref_dir = $ref_dir."/" unless rindex($ref_dir, "/") == length($ref_dir) - 1;
open GTF, "<", $gtf or die "cannot open gtf file: $!";
open OUT, ">", $out or die;
while(<GTF>){
chomp;
next if /^#/;
my @line = split /\t/;
if ( ($if_chr == 1 and $line[0] ne "chr1") or $line[0] eq "chrM" ){
last;
}
my @atr = split '; ', $line[8];
if($pre_gene ne $atr[0] and $pre_gene ne ''){
&split_transcript(@gene_anno);
#&split_transcript_all(@gene_anno);
@gene_anno = ();
#print "$line[0]\t$pre_gene\n";
}
push @gene_anno, $_;
$pre_gene = $atr[0];
}
#for my $chr(@chr){
# for my $gene(keys %{$chr_$gene_trsc_exon{$chr}}){
# for my $trsc(keys %{$chr_$gene_trsc_exon{$chr}{$gene}}){
# for $exon(@{$chr_$gene_trsc_exon{$chr}{$gene}{$trsc}}){
# print OUT "$chr\t$gene\t$trsc\t@$exon\n";
# }
# }
# }
#}
for my $chromo(@chr){
open CHR, "<", $ref_dir."$chromo.fa" or open CHR, "<", $ref_dir."$chromo.fasta" or die "cannot open the chr fasta file $chromo: $!";
my $uni_seq = 0;
my $chr_seq;
while(<CHR>){
chomp;
if(/^>/ and $uni_seq == 0){
$uni_seq = 1;
}elsif(/^>/){
die "There are more than one sequence in $chromo file. Please check!";
}else{
$chr_seq .= $_;
}
}
my ($count, $gene_num, $inter_num, $circ_id);
my @intron_gene;
my $total_gene = keys %{$chr_gene_trsc_exon{$chromo}};
print $chromo."\t".$total_gene."\n";
##generating exon circ
GEN: for my $gene(keys %{$chr_gene_trsc_exon{$chromo}}){
$count += 1;
if ($count > ($total_gene * 0.8)){
push (@intron_gene, $gene);
next GEN;
}
my @trscs = keys (%{$chr_gene_trsc_exon{$chromo}{$gene}});
my $trsc_rand = int(rand($#trscs+1));
my $trsc = $trscs[$trsc_rand];
my %ss_exon_id;
my ($cRNA_seq, $cRNA_seq2);
my $trsc_seq;
my $if_cRNA = int(rand(2)); #the probability of cRNA generated from this transcrpt
my $if_linear = int(rand(2));
for my $i(0 .. $#{$chr_gene_trsc_exon{$chromo}{$gene}{$trsc}}){
my $exon = $chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$i];
my $exon_seq;
if($$exon[2] eq '+'){
$exon_seq = substr($chr_seq, $$exon[0]-1, $$exon[1]-$$exon[0]+1);
}else{
$exon_seq = &comp_rev(substr($chr_seq, $$exon[0]-1, $$exon[1]-$$exon[0]+1));
}
push @$exon, $exon_seq;
push @$exon, substr($chr_seq, $$exon[0]-3, 2);
push @$exon, substr($chr_seq, $$exon[1], 2);
$trsc_seq .= $$exon[-3];
if ( $if_cRNA == 1 and ($$exon[1]-$$exon[0]+1>20 and $$exon[1]-$$exon[0]+1<2000) and ( ($$exon[2] eq '-' and $$exon[-2] =~ /AC/i and $$exon[-1] =~ /CT/i) or ($$exon[2] eq '+' and $$exon[-2] =~ /AG/i and $$exon[-1] =~ /GT/i) ) ){
#$rand_exon1 ++;
#$rand_num1{$rand_exon1} = $i;
$ss_exon_id{$i} = 1;
}
}
&simulate_reads2( $rand_mode2, $trsc_seq, $coverage2 ) if ($if_linear == 1); #length($trsc_seq) > $insert_length and
if( $if_cRNA == 1 and scalar(keys %ss_exon_id)>=3 ){ #and $rand_exon2>=1
my @qualified_exon_sort = sort {$a <=> $b} (keys %ss_exon_id);
my ($bingo_num1, $bingo_num2, $bingo_num3);
my (%p_loci1, %p_loci2, $total_p1, $total_p2, $accu_p1, $accu_p2);
for my $qualified_exon($qualified_exon_sort[0] .. $qualified_exon_sort[-1]){
next GEN if !exists $ss_exon_id{$qualified_exon};
}
my $pre_loci1 = int($#qualified_exon_sort/2)-2*log(@qualified_exon_sort)/log(10);
my $pre_loci2 = int($#qualified_exon_sort/2)+2*log(@qualified_exon_sort)/log(10)-1; ########################################### add -1
for my $i(0 .. int($#qualified_exon_sort/2)-$exon_skipping){
$p_loci1{$i} = 1/(($i-$pre_loci1)**2+.01);
$total_p1 += $p_loci1{$i};
}
for my $i(int($#qualified_exon_sort/2) .. $#qualified_exon_sort){ ########################################### delete +1
$p_loci2{$i} = 1/(($i-$pre_loci2)**2+.01);
$total_p2 += $p_loci2{$i};
}
my $dice_loci1 = rand(1);
for my $i(0 .. int($#qualified_exon_sort/2)-$exon_skipping){
my $accu_pre = $accu_p1;
$accu_p1 += $p_loci1{$i}/$total_p1;
if ($dice_loci1 > $accu_pre and $dice_loci1 <= $accu_p1){
$bingo_num1 = $i;
}
}
if(!defined $bingo_num1){
$bingo_num1 = int($#qualified_exon_sort/2)-1;
print "1!!$dice_loci1\t", scalar(@qualified_exon_sort),"\n";
}
my $dice_loci2 = rand(1);
for my $i(int($#qualified_exon_sort/2) .. $#qualified_exon_sort){
my $accu_pre = $accu_p2;
$accu_p2 += $p_loci2{$i}/$total_p2;
if ($dice_loci2 > $accu_pre and $dice_loci2 <= $accu_p2){
$bingo_num2 = $i;
}
}
if(!defined $bingo_num2){
$bingo_num2 = int($#qualified_exon_sort/2)+1;
print "2!!$dice_loci2\t", scalar(@qualified_exon_sort),"\n";
}
next GEN if $bingo_num1>$bingo_num2; ########################################### add this to prevent error
if(int(rand($exon_skipping + 1)) == 1){ #should have at least three exons and $rand_num1{$bingo_num1} <= $rand_num1{$bingo_num2}-2
$bingo_num3 = int(rand($bingo_num2-$bingo_num1-1)+$bingo_num1+1);
if ($bingo_num3 < $bingo_num2 and $bingo_num3 > $bingo_num1){
for my $j( $qualified_exon_sort[$bingo_num1] .. $qualified_exon_sort[$bingo_num2] ){
my $exon = $chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$j];
$cRNA_seq .= $$exon[-3];
$cRNA_seq2 .= $$exon[-3] unless $j == $qualified_exon_sort[$bingo_num3];
}
next GEN if length($cRNA_seq2) <100 or length($cRNA_seq2) >4000;
next GEN if length($cRNA_seq) <100 or length($cRNA_seq) >4000;
if ($chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$qualified_exon_sort[$bingo_num1]][0] < $chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$qualified_exon_sort[$bingo_num2]][1]){
print OUT "$chromo\t$gene\t$trsc\t", $chromo, ":" , $chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$qualified_exon_sort[$bingo_num1]][0], "|", $chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$qualified_exon_sort[$bingo_num2]][1], "\texon", "\nisoform1_", length($cRNA_seq), "\t";
$circ_id = $chromo.":".$chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$qualified_exon_sort[$bingo_num1]][0]."|".$chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$qualified_exon_sort[$bingo_num2]][1];
}elsif($chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$qualified_exon_sort[$bingo_num1]][1] > $chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$qualified_exon_sort[$bingo_num2]][0]){
print OUT "$chromo\t$gene\t$trsc\t", $chromo, ":" , $chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$qualified_exon_sort[$bingo_num2]][0], "|", $chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$qualified_exon_sort[$bingo_num1]][1], "\texon", "\nisoform1_", length($cRNA_seq), "\t";
$circ_id = $chromo.":".$chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$qualified_exon_sort[$bingo_num2]][0]."|".$chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$qualified_exon_sort[$bingo_num1]][1];
}else{
next GEN;
}
for my $j( $qualified_exon_sort[$bingo_num1] .. $qualified_exon_sort[$bingo_num2] ){
my $exon = $chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$j];
print OUT "$$exon[0]:$$exon[1]!$$exon[2],";
}
print OUT "\nisoform2_", length($cRNA_seq2), "\t";
for my $j( $qualified_exon_sort[$bingo_num1] .. $qualified_exon_sort[$bingo_num2] ){
my $exon = $chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$j];
print OUT "$$exon[0]:$$exon[1]!$$exon[2]," unless $j == $qualified_exon_sort[$bingo_num3];
}
print OUT "\n";
&simulate_reads( $rand_mode, $cRNA_seq, $coverage*$psi/100, $circ_id );
&simulate_reads( $rand_mode, $cRNA_seq2, $coverage*(100-$psi)/100, $circ_id );
$sim_total++;
}else{
#print "!!!$bingo_num1\t$bingo_num2\t$bingo_num3\n";
}
}else{ #should have at least two exons and $rand_num1{$bingo_num1} <= $rand_num1{$bingo_num2}-1
for my $j( $qualified_exon_sort[$bingo_num1].. $qualified_exon_sort[$bingo_num2] ){
my $exon = $chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$j];
$cRNA_seq .= $$exon[-3];
#$cRNA_seq2 .= $$exon[-3] unless $j == $rand_num1{$bingo_num3};
}
next GEN if length($cRNA_seq) < 100 or length($cRNA_seq) >4000;
if ($chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$qualified_exon_sort[$bingo_num1]][0] < $chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$qualified_exon_sort[$bingo_num2]][1]){
print OUT "$chromo\t$gene\t$trsc\t", $chromo, ":" , $chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$qualified_exon_sort[$bingo_num1]][0], "|", $chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$qualified_exon_sort[$bingo_num2]][1], "\texon", "\nisoform1_", length($cRNA_seq), "\t";
$circ_id = $chromo.":".$chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$qualified_exon_sort[$bingo_num1]][0]."|".$chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$qualified_exon_sort[$bingo_num2]][1];
}elsif($chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$qualified_exon_sort[$bingo_num1]][1] > $chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$qualified_exon_sort[$bingo_num2]][0]){
print OUT "$chromo\t$gene\t$trsc\t", $chromo, ":" , $chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$qualified_exon_sort[$bingo_num2]][0], "|", $chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$qualified_exon_sort[$bingo_num1]][1], "\texon", "\nisoform1_", length($cRNA_seq), "\t";
$circ_id = $chromo.":".$chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$qualified_exon_sort[$bingo_num2]][0]."|".$chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$qualified_exon_sort[$bingo_num1]][1];
}else{
next GEN;
}
for my $j( $qualified_exon_sort[$bingo_num1] .. $qualified_exon_sort[$bingo_num2] ){
my $exon = $chr_gene_trsc_exon{$chromo}{$gene}{$trsc}[$j];
print OUT "$$exon[0]:$$exon[1]!$$exon[2],";
}
print OUT "\n";
&simulate_reads( $rand_mode, $cRNA_seq, $coverage, $circ_id );
#&simulate_reads( $rand_mode, $cRNA_seq2, $coverage );
$sim_total++;
}
}
}
##generating intron circRNA
GEN_ALL: for my $gene(keys %{$chr_gene_trsc_all{$chromo}}){
#print $gene."\n";
next unless grep /^$gene$/, @intron_gene;###next unless $gene ~~ @intron_gene;
my ($cRNA_seq, $cRNA_seq2, $trsc_seq, %ss_element_id);
my @trscs = keys (%{$chr_gene_trsc_all{$chromo}{$gene}});
my $trsc_rand = int(rand($#trscs+1));
my $trsc = $trscs[$trsc_rand];
my $if_cRNA = int(rand(2));
### single intron circRNA
$gene_num += 1;
if ($gene_num < ($#intron_gene * 0.05)){
if ( $if_cRNA == 1 ) {
my $i = int(rand($#{$chr_gene_trsc_intron{$chromo}{$gene}{$trsc}}));
my $element = $chr_gene_trsc_intron{$chromo}{$gene}{$trsc}[$i];
next GEN_ALL if $$element[1]-$$element[0]>20 and $$element[1]-$$element[0]<4000;
if($$element[2] eq '+'){
$cRNA_seq = substr($chr_seq, $$element[0]-1, $$element[1]-$$element[0]+1);
}else{
$cRNA_seq = &comp_rev(substr($chr_seq, $$element[0]-1, $$element[1]-$$element[0]+1));
}
next GEN_ALL if length($cRNA_seq) < 100 or length($cRNA_seq) >4000;
print OUT "$chromo\t$gene\t$trsc\t", $chromo, ":" , $$element[0], "|", $$element[1], "\tintron", "\nisoform1_", length($cRNA_seq), "\t";
$circ_id = $chromo.":".$$element[0]."|".$$element[1];
print OUT "\n";
&simulate_reads( $rand_mode, $cRNA_seq, $coverage*$psi/100, $circ_id );
$sim_total++;
next GEN_ALL;
} else {
next GEN_ALL;
}
}
for my $i(0 .. $#{$chr_gene_trsc_all{$chromo}{$gene}{$trsc}}){
my $element = $chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$i];
my $element_seq;
if($$element[2] eq '+'){
$element_seq = substr($chr_seq, $$element[0]-1, $$element[1]-$$element[0]+1);
}else{
$element_seq = &comp_rev(substr($chr_seq, $$element[0]-1, $$element[1]-$$element[0]+1));
}
push @$element, $element_seq;
push @$element, substr($chr_seq, $$element[0]-3, 2);
push @$element, substr($chr_seq, $$element[1], 2);
$trsc_seq .= $$element[-3];
if ( $if_cRNA == 1 and ($$element[1]-$$element[0]+1>20 and $$element[1]-$$element[0]+1<2000) ){
$ss_element_id{$i} = 1;
}
}
if( $if_cRNA == 1 and scalar(keys %ss_element_id)>=3 ){ #and $rand_element2>=1
my @qualified_element_sort = sort {$a <=> $b} (keys %ss_element_id);
my ($bingo_all_num1, $bingo_all_num2, $bingo_all_num3);
my (%p_all_loci1, %p_all_loci2, $total_all_p1, $total_all_p2, $accu_all_p1, $accu_all_p2);
for my $qualified_element($qualified_element_sort[0] .. $qualified_element_sort[-1]){
next GEN_ALL if !exists $ss_element_id{$qualified_element};
}
my $pre_all_loci1 = int($#qualified_element_sort/2)-2*log(@qualified_element_sort)/log(10);
my $pre_all_loci2 = int($#qualified_element_sort/2)+2*log(@qualified_element_sort)/log(10)-1; ########################################### add -1
for my $i(0 .. int($#qualified_element_sort/2)-$exon_skipping){
$p_all_loci1{$i} = 1/(($i-$pre_all_loci1)**2+.01);
$total_all_p1 += $p_all_loci1{$i};
}
for my $i(int($#qualified_element_sort/2) .. $#qualified_element_sort){ ########################################### delete +1
$p_all_loci2{$i} = 1/(($i-$pre_all_loci2)**2+.01);
$total_all_p2 += $p_all_loci2{$i};
}
my $dice_all_loci1 = rand(1);
for my $i(0 .. int($#qualified_element_sort/2)-$exon_skipping){
my $accu_all_pre = $accu_all_p1;
$accu_all_p1 += $p_all_loci1{$i}/$total_all_p1;
if ($dice_all_loci1 > $accu_all_pre and $dice_all_loci1 <= $accu_all_p1){
$bingo_all_num1 = $i;
}
}
if(!defined $bingo_all_num1){
$bingo_all_num1 = int($#qualified_element_sort/2)-1;
print "1!!$dice_all_loci1\t", scalar(@qualified_element_sort),"\n";
}
my $dice_all_loci2 = rand(1);
for my $i(int($#qualified_element_sort/2) .. $#qualified_element_sort){
my $accu_all_pre = $accu_all_p2;
$accu_all_p2 += $p_all_loci2{$i}/$total_all_p2;
if ($dice_all_loci2 > $accu_all_pre and $dice_all_loci2 <= $accu_all_p2){
$bingo_all_num2 = $i;
}
}
if(!defined $bingo_all_num2){
$bingo_all_num2 = int($#qualified_element_sort/2)+1;
print "2!!$dice_all_loci2\t", scalar(@qualified_element_sort),"\n";
}
next GEN_ALL if $bingo_all_num1>$bingo_all_num2; ########################################### add this to prevent error
if($exon_skipping == 1){ #should have at least three elements and $rand_num1{$bingo_all_num1} <= $rand_num1{$bingo_all_num2}-2
$bingo_all_num3 = int(rand($bingo_all_num2-$bingo_all_num1-1)+$bingo_all_num1+1);
if ($bingo_all_num3 < $bingo_all_num2 and $bingo_all_num3 > $bingo_all_num1){
for my $j( $qualified_element_sort[$bingo_all_num1] .. $qualified_element_sort[$bingo_all_num2] ){
my $element = $chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$j];
$cRNA_seq .= $$element[-3];
$cRNA_seq2 .= $$element[-3] unless $j == $qualified_element_sort[$bingo_all_num3];
}
next GEN_ALL if length($cRNA_seq2) <100 or length($cRNA_seq2) >4000;
next GEN_ALL if length($cRNA_seq) <100 or length($cRNA_seq) >4000; # or length($cRNA_seq) > 850;
if ($chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$qualified_element_sort[$bingo_all_num1]][0] < $chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$qualified_element_sort[$bingo_all_num2]][1]){
print OUT "$chromo\t$gene\t$trsc\t", $chromo, ":" , $chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$qualified_element_sort[$bingo_all_num1]][0], "|", $chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$qualified_element_sort[$bingo_all_num2]][1], "\tintron", "\nisoform1_", length($cRNA_seq), "\t";
$circ_id = $chromo.":".$chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$qualified_element_sort[$bingo_all_num1]][0]."|".$chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$qualified_element_sort[$bingo_all_num2]][1];
}elsif($chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$qualified_element_sort[$bingo_all_num1]][1] > $chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$qualified_element_sort[$bingo_all_num2]][0]){
print OUT "$chromo\t$gene\t$trsc\t", $chromo, ":" , $chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$qualified_element_sort[$bingo_all_num2]][0], "|", $chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$qualified_element_sort[$bingo_all_num1]][1], "\tintron", "\nisoform1_", length($cRNA_seq), "\t";
$circ_id = $chromo.":".$chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$qualified_element_sort[$bingo_all_num2]][0]."|".$chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$qualified_element_sort[$bingo_all_num1]][1];
}else{
next GEN_ALL;
}
for my $j( $qualified_element_sort[$bingo_all_num1] .. $qualified_element_sort[$bingo_all_num2] ){
my $element = $chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$j];
print OUT "$$element[0]:$$element[1]!$$element[2],";
}
print OUT "\nisoform2_", length($cRNA_seq2), "\t";
for my $j( $qualified_element_sort[$bingo_all_num1] .. $qualified_element_sort[$bingo_all_num2] ){
my $element = $chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$j];
print OUT "$$element[0]:$$element[1]!$$element[2]," unless $j == $qualified_element_sort[$bingo_all_num3];
}
print OUT "\n";
&simulate_reads( $rand_mode, $cRNA_seq, $coverage*$psi/100, $circ_id );
&simulate_reads( $rand_mode, $cRNA_seq2, $coverage*(100-$psi)/100, $circ_id );
$sim_total++;
}else{
print "!!!$bingo_all_num1\t$bingo_all_num2\t$bingo_all_num3\n";
}
}elsif($exon_skipping == 0){ #should have at least two elements and $rand_num1{$bingo_all_num1} <= $rand_num1{$bingo_all_num2}-1
for my $j( $qualified_element_sort[$bingo_all_num1].. $qualified_element_sort[$bingo_all_num2] ){
my $element = $chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$j];
$cRNA_seq .= $$element[-3];
#$cRNA_seq2 .= $$element[-3] unless $j == $rand_num1{$bingo_all_num3};
}
next GEN_ALL if length($cRNA_seq) < 100 or length($cRNA_seq) >4000;
if ($chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$qualified_element_sort[$bingo_all_num1]][0] < $chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$qualified_element_sort[$bingo_all_num2]][1]){
print OUT "$chromo\t$gene\t$trsc\t", $chromo, ":" , $chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$qualified_element_sort[$bingo_all_num1]][0], "|", $chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$qualified_element_sort[$bingo_all_num2]][1], "\tintron", "\nisoform1_", length($cRNA_seq), "\t";
$circ_id = $chromo.":".$chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$qualified_element_sort[$bingo_all_num1]][0]."|".$chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$qualified_element_sort[$bingo_all_num2]][1];
}elsif($chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$qualified_element_sort[$bingo_all_num1]][1] > $chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$qualified_element_sort[$bingo_all_num2]][0]){
print OUT "$chromo\t$gene\t$trsc\t", $chromo, ":" , $chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$qualified_element_sort[$bingo_all_num2]][0], "|", $chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$qualified_element_sort[$bingo_all_num1]][1], "\tintron", "\nisoform1_", length($cRNA_seq), "\t";
$circ_id = $chromo.":".$chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$qualified_element_sort[$bingo_all_num2]][0]."|".$chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$qualified_element_sort[$bingo_all_num1]][1];
}else{
next GEN_ALL;
}
for my $j( $qualified_element_sort[$bingo_all_num1] .. $qualified_element_sort[$bingo_all_num2] ){
my $element = $chr_gene_trsc_all{$chromo}{$gene}{$trsc}[$j];
print OUT "$$element[0]:$$element[1]!$$element[2],";
}
print OUT "\n";
&simulate_reads( $rand_mode, $cRNA_seq, $coverage, $circ_id );
#&simulate_reads( $rand_mode, $cRNA_seq2, $coverage );
$sim_total++;
}
}
}
###generating intergenic circRNA
INTER: for my $i(0 .. $#{$chr_inter{$chromo}}){
my $region = $chr_inter{$chromo}[$i];
my $inter_total_num = $#intron_gene * 0.1;
$inter_num += 1;
next INTER if $inter_num > $inter_total_num;
my ($if_cRNA, $pre_length, $inter_circ_start, $inter_circ_end, $inter_circ_strand, $cRNA_seq);
$if_cRNA = int(rand(2));
next INTER if $if_cRNA == 0;
$pre_length = length($$region[1] - $$region[0] + 1);
$inter_circ_start = int(rand($pre_length * 0.5)) + $$region[0];
$inter_circ_end = $inter_circ_start + 100 + int(rand(3800));
if (int(rand(2)) == 1){
$inter_circ_strand = "+";
} else {
$inter_circ_strand = "-";
}
if($inter_circ_strand eq '+'){
$cRNA_seq = substr($chr_seq, $inter_circ_start-1, $inter_circ_end-$inter_circ_start+1);
}else{
$cRNA_seq = &comp_rev(substr($chr_seq, $inter_circ_start-1, $inter_circ_end-$inter_circ_start+1));
}
print OUT "$chromo\t", $chromo, ":" , $inter_circ_start, "|", $inter_circ_end, "\tintergenic", "\nisoform1_", length($cRNA_seq), "\t", $inter_circ_start, ":", $inter_circ_end, "!", $inter_circ_strand, ",";
$circ_id = $chromo.":".$inter_circ_start."|".$inter_circ_end;
print OUT "\n";
&simulate_reads( $rand_mode, $cRNA_seq, $coverage*$psi/100, $circ_id );
$sim_total++;
}
}
print OUT "!!total: $sim_total\n";
print "circRNA totally simulated: $sim_total\n";
sub simulate_reads2{
my $mode = shift @_;
my $trsc_coverage;
my $seq_length = length($_[0]);
if ($mode == 1){
$trsc_coverage = $_[1];
}else{
$trsc_coverage = rand($_[1]+1);
}
my ($read_num, undef) = sort{$b <=> $a}(int( $seq_length * $trsc_coverage / $read_length / 2 ),1);
my $err_num = int( $seq_length * $trsc_coverage * $seq_err / 100 );
my %err_read;
for (1 .. $err_num){
my $err_loci = int( rand( (($read_num)*2)+1 ) );
$err_read{$err_loci} ++;
}
for my $x( 1 .. $read_num ){
my $ins_len_rand = &insert_length_calculation_normal(rand(1), rand(1), rand(1)); #insert length can be simulated later
#my $start_loci = int( rand($seq_length - $ins_len_rand - $read_length) );
next if $ins_len_rand < 0 or $ins_len_rand >= $seq_length-$read_length;
my ($start_loci, $start_loci2);
$start_loci = int( rand($seq_length-$ins_len_rand-$read_length+1) );
$start_loci2 = $start_loci + $ins_len_rand; # - $read_length;
next if $start_loci2 > $seq_length-$read_length;
$seqID ++;
my $if_1st = int(rand(2));
my ($seq1, $seq2);
if ($if_1st == 1){
$seq1 = substr( $_[0], $start_loci, $read_length );
$seq2 = &comp_rev( substr( $_[0], $start_loci2, $read_length ) );
}else{
$seq2 = substr( $_[0], $start_loci, $read_length );
$seq1 = &comp_rev( substr( $_[0], $start_loci2, $read_length ) );
}
die "$seq1" if length($seq1) != $read_length;
die "$seq2" if length($seq2) != $read_length;
my @errs1;
for (1 .. $err_read{$x * 2 - 1}){
my $err_loci = int( rand($read_length) );
redo if $err_loci ~~ @errs1;
push @errs1, $err_loci;
my $ori_base = substr( $seq1, $err_loci, 1 );
substr( $seq1, $err_loci, 1 ) = &simulate_seq_error($ori_base);
}
my @errs2;
for (1 .. $err_read{$x * 2}){
my $err_loci = int( rand($read_length) );
redo if $err_loci ~~ @errs2;
push @errs2, $err_loci;
my $ori_base = substr( $seq2, $err_loci, 1 );
substr( $seq2, $err_loci, 1 ) = &simulate_seq_error($ori_base);
}
print FQ1 '@simulate:'."$seqID/1 length=$read_length\n";
print FQ2 '@simulate:'."$seqID/2 length=$read_length\n";
print FQ1 "$seq1\n";
print FQ2 "$seq2\n";
print FQ1 "+\n";
print FQ2 "+\n";
print FQ1 ("!" x $read_length) . "\n";
print FQ2 ("!" x $read_length) . "\n";
}
}
sub insert_length_calculation_linear{
if($_[0]<=0.5){
my $x = int(($_[0]*2)**.5*$insert_length);
}else{
my $x = int((2-(2-2*$_[0])**.5)*$insert_length);
}
}
sub insert_length_calculation_normal{
my $y;
if($_[2] < $perc_minor/100){
$y = sqrt(-2*log($_[0]))*cos(2*$pi*$_[1])*$sigma2+$insert_length2;
}else{
$y = sqrt(-2*log($_[0]))*cos(2*$pi*$_[1])*$sigma+$insert_length;
}
}
sub simulate_reads{
my $mode = shift @_;
my $seq_length = length($_[0]);
my $cRNA_coverage;
my $junc_read = ();
my $circ_id = $_[-1];
#my $seq4substr;
if ($mode == 1){
$cRNA_coverage = $_[1];
}else{
$cRNA_coverage = rand($_[1]+1);
}
my $seq4substr = $_[0] x 12;
my $read_num = int( $seq_length * $cRNA_coverage / $read_length / 2 );
my $err_num = int( $seq_length * $cRNA_coverage * $seq_err / 100 );
my %err_read;
for (1 .. $err_num){
my $err_loci = int( rand( ($read_num)*2 ) +1);
$err_read{$err_loci} ++;
}
for my $x( 1 .. $read_num ){
my $ins_len_rand = &insert_length_calculation_normal(rand(1), rand(1), rand(1)); #insert length can be simulated later
next if $ins_len_rand < 0; #( or $ins_len_rand + $read_length > $seq_length );
my ($start_loci, $start_loci2);
$start_loci = int( rand($seq_length) );
$start_loci2 = $start_loci + $ins_len_rand; # - $read_length;
$seqID ++;
my $if_1st = int( rand(2) );
my ($seq1, $seq2);
$junc_read .= $seqID."," if ($start_loci > ($seq_length - $read_length)) and ($start_loci < $seq_length);
for my $times (1 .. 9) {
$junc_read .= $seqID."," if $start_loci2 > (($seq_length * $times) - $read_length) and $start_loci2 < ($seq_length * $times);
}
if ($if_1st == 1){
$seq1 = substr( $seq4substr, $start_loci, $read_length );
$seq2 = &comp_rev( substr( $seq4substr, $start_loci2, $read_length ) );
}else{
$seq2 = substr( $seq4substr, $start_loci, $read_length );
$seq1 = &comp_rev( substr( $seq4substr, $start_loci2, $read_length ) );
}
die "$seq2" if length($seq2) != $read_length;
die "$seq1" if length($seq1) != $read_length;
my @errs1;
for (1 .. $err_read{$x * 2 - 1}){
my $err_loci = int( rand($read_length) );
redo if $err_loci ~~ @errs1;
push @errs1, $err_loci;
my $ori_base = substr( $seq1, $err_loci, 1 );
substr( $seq1, $err_loci, 1) = &simulate_seq_error($ori_base);
}
my @errs2;
for (1 .. $err_read{$x * 2}){
my $err_loci = int( rand($read_length) );
redo if $err_loci ~~ @errs2;
push @errs2, $err_loci;
my $ori_base = substr( $seq2, $err_loci, 1 );
substr( $seq2, $err_loci, 1) = &simulate_seq_error($ori_base);
}
print FQ1 '@simulate:'."$seqID/1 length=$read_length\n";
print FQ2 '@simulate:'."$seqID/2 length=$read_length\n";
print FQ1 "$seq1\n";
print FQ2 "$seq2\n";
print FQ1 "+\n";
print FQ2 "+\n";
print FQ1 ("!" x $read_length) . "\n";
print FQ2 ("!" x $read_length) . "\n";
print OUT ">\t$x\t$seqID\n";
print CIRCREAD "$circ_id\tsimulate:"."$seqID\n";
if($if_1st == 1){
print OUT "**\t1\n" if ($start_loci >= $seq_length-$read_length+20 and $start_loci <= $seq_length-20);
print OUT "**\t2\n" if ($start_loci2%$seq_length >= $seq_length-$read_length+20 and $start_loci2%$seq_length <= $seq_length-20);
}else{
print OUT "**\t1\n" if ($start_loci2%$seq_length >= $seq_length-$read_length+20 and $start_loci2%$seq_length <= $seq_length-20);
print OUT "**\t2\n" if ($start_loci >= $seq_length-$read_length+20 and $start_loci <= $seq_length-20);
}
}
my (@junc, $tmp, %exist_junc, $junc_read_id);
if ($junc_read =~ /\d+/){
@junc = split /,/,$junc_read;
for $tmp (@junc){
$exist_junc{$tmp} = 1;
}
for (sort keys %exist_junc){
$junc_read_id .= "simulate:".$_.",";
}
print CIRCREAD "$circ_id\tbsj:"."$junc_read_id\n";
}
}
sub simulate_seq_error{
my $ori_base = $_[0];
my @base = ('A', 'T', 'C', 'G');
my $err_base_index;
for my $i( 0 .. $#base ){
if ($base[$i] =~ /$ori_base/i){
while(1){
$err_base_index = int(rand(4));
last unless $err_base_index == $i;
}
last;
}
}
$base[$err_base_index];
}
sub comp_rev{
my $seq = reverse($_[0]);
$seq =~ s/[Aa]/X/g;
$seq =~ s/[Tt]/A/g;
$seq =~ s/X/T/g;
$seq =~ s/[Cc]/Y/g;
$seq =~ s/[Gg]/C/g;
$seq =~ s/Y/G/g;
$seq;
}
sub split_transcript{
my ($count, $intron_end, $intron_start, $intron_strand, $inter_start, $inter_end, $transcript, %trans_exist, %chr_inter_exist, %intron);
for (@_){
my @line = split /\t/;
next if $line[0] =~ /\_/;
if($line[2] eq 'exon'){
my @atr = split ('; ', $line[8], 3);
if ($atr[1] =~ /transcript_id \"(\S+?)\"/){
##exon
push @{$chr_gene_trsc_exon{$line[0]}{$atr[0]}{$1}}, [ $line[3], $line[4], $line[6] ];
##exon and intron
unless ($intron{$1}) {
$intron{$1} = 1;
$intron_start = $line[4];
push @{$chr_gene_trsc_all{$line[0]}{$atr[0]}{$1}}, [ $line[3], $line[4], $line[6] ];
} else {
$intron_end = $line[3];
$intron_strand = $line[6];
push @{$chr_gene_trsc_all{$line[0]}{$atr[0]}{$1}}, [ $intron_start, $intron_end, $intron_strand ];
push @{$chr_gene_trsc_intron{$line[0]}{$atr[0]}{$1}}, [ $intron_start, $intron_end, $intron_strand ];
$intron_start = $line[4];
push @{$chr_gene_trsc_all{$line[0]}{$atr[0]}{$1}}, [ $line[3], $line[4], $line[6] ];
}
#$trans_exist{$1} = 1 and $intron_start = () unless $chr_gene_trsc_all{$line[0]}{$atr[0]}{$1};
}else{
print "error: no transcript_id found for $atr[0]!\n";
}
}
##intergenic
if($line[2] eq 'transcript'){
$chr_inter_exist{$line[0]} = 1 and $inter_start = () unless $chr_inter_exist{$line[0]};
unless ($inter_start){
$inter_start = $line[4];
} else {
$inter_end = $line[3];
push @{$chr_inter{$line[0]}}, [ $inter_start, $inter_end ];
$inter_start = $line[4];
}
}
}
#print $_[0]."\n";
my @line2 = split (/\t/, $_[0], 2);
push @chr, $line2[0] unless $line2[0] ~~ @chr;
}