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tea.pl
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#!/usr/bin/perl
use strict;
no strict "refs";
use Getopt::Std;
use File::Basename;
use IO::File;
use IO::Compress::Gzip qw(gzip $GzipError);
use List::Util qw(min max sum);
my $cmd = basename($0);
# user-defined variables
#my $blast_path = "/home/el114/download/ncbi-blast-2.2.26+/bin";
#my $blast_path = "/bitools/BLAST/ncbi-blast-2.2.27+/bin";
#my $bwa = "/bitools/BWA/bwa-0.7.5a/bwa";
#my $samtools = "/bitools/SAMTools/samtools-0.1.19/samtools";
#my $assembler = "/bitools/CAP3/CAP3_07-10-15/cap3";
my $line;
my %hash = ();
open my $f, "<", "conf_file";
while($line=<$f>){
my @val = split('=', $line);
$val[0] =~ s/^\s+//;
$val[0] =~ s/\s+$//;
$val[1] =~ s/^\s+//;
$val[1] =~ s/\s+$//;
$hash{$val[0]} = $val[1];
}
close $f;
my $blast_path = "$hash{'blast_home'}";
my $bwa = "$hash{'bwa_home'}"; $bwa = "$bwa/bwa";
my $samtools = "$hash{'samtools_home'}"; $samtools = "$samtools/samtools";
my $assembler = "$hash{'cap3_home'}"; $assembler = "$assembler/cap3";
my $bzip2 = "bzip2";
my $usage = qq{
Usage: $cmd <command> [options]
Command: tea generate a shell script to run tea
bam2fq generate fastq files from a bam file
cbam extract the soft-clipping positions and make a bam only with the clipped reads
bamram extract the coordinates of rams and generate a bam only with rams
isize extract the distribution of fragment sizes and write it to the standard output
cisize format the isize and read length information from meerkat output
contig generate contigs of ram mates and clipped sequences
map_consensus <infile> <family:L1|Alu|PABL_A-INT> <map_subfmaily: 1|0>
rmasker_coordinates <input repeat masker file: *.gz> <1st record line#> <verbose: 1|0>
rmasker_coordinates_strand <input repeat masker file: *.gz> <1st record line#> <verbose: 1|0>
ram extract the coordinates of rams per te type
sram separte rams per each repeat type
mapcnt count the mapped reads for each sequence
rmdup remove PCR duplicates in a bam file with unmapped reads
};
my $log_dir = ".";
if (@ARGV < 1) { die $usage }
$cmd = shift;
my @functions = qw(tea bam2fq cbam ram bamram isize cisize sram contig map_consensus make_subfamilyseq
rmasker_coordinates mapcnt rmdup rmasker_coordinates_strand);
if ($cmd eq "map_consensus") {
my ($infile, $family, $map_subfamily) = @ARGV;
&map_consensus($infile, $family, $map_subfamily, 0);
exit(1);
}
if ($cmd eq "make_subfamilyseq") {
my ($family) = @ARGV;
&make_subfamilyseq($family);
exit(1);
}
if (grep {$_ eq $cmd} @functions) { &$cmd(); }
else { die $usage; }
sub tea
{
my $usage = qq{
Usage: tea.pl [options] <sample id>
generate a shell script to perform the following steps
1. procss input files--sorting, indexing, an formatting
disc.sorted.bam
cl.disc.sorted.bam (if no.clipped=F)
cl.sorted.bam (if exogenous=T)
isinfo (isize and read length)
2. generate fastq files
3. mapping
4. generate ram.bam and ram.bz2
5. cbam prep
6. run (rid | rid.te | rid.te.primate)
7. run (germline | te.primate.germline)
Options: -d STR base dir (.)
-p INT number of threads for bwa alignment
-P STR tea base dir to add to \$PATH
-F skip the step four, cbam generation (no)
-g STR a bam file to extract clipped reads
-l INT bwa aln seed lenegh (40)
-k INT bwa aln maximum differences in the seed (2)
-n INT bwa aln max #diff (int) or missing prob under 0.02 err rate (floot) (3)
-r STR sequence assembly (.fasta)
-f family-wise merge of rams (no)
-R STR assembly symbol (ra and va for endogenoeus and exogenous)
-s STR scratch or temp space to save the intermediate mapping files (/tmp)
-c STR a comma separated chromosome list or ref
[hg18|hg19|panTro3|ponAbe2|rheMac2] (hg18)
-a INT start from the given step
-x exogenous (no)
-S no clipped reads and cl.sorted.bam
-C cleaning up the intermediate files such as fastq, ra.bam, etc. (no)
-M INT minimum ram
-o also reporting clusters with only one side ram cluster (no)
-j INT jittering or allowed gap in extracting clipped positions (2)
-e STR rid execution function (rid | rid.te | rid.te.primate) (rid)
-t contig generation (no)
### LSF parameters
-m LSF job for each chr (no)
-L STR LSF log dir (./log)
-q STR LSF queue (long)
-b STR LSF estimated run time (72:00)
};
my %opts = ();
getopts("hd:g:l:k:n:r:s:c:Smq:GP:a:p:xR:j:oM:fFie:tb:", \%opts);
if (@ARGV < 1 || exists($opts{h})) { die $usage };
my $sample = shift(@ARGV);
# set defaults
my $bdir = ".";
my $bamf = "";
my $logdir = "./log";
my $l = 40;
my $k = 2;
my $n = 3;
my $ref = "hg18";
my $no_clipped = 0;
my $parallel = 0;
my $tea = "";
my $scratch = "/tmp";
my $q = "long";
my $sortmem = "2000000000";
my $clean = 0;
my $step = 1;
my $nproc = 1;
my $exogenous = 0;
my $minram = 3;
my $oneside_ram = 0;
my $jittering = 2;
my $rasym = "ra";
my $family = 0;
my $skip_cbam = 0;
my $exec = "rid";
my $contig = 0;
my $time = "72:00";
$bdir = $opts{d} if (defined($opts{d}));
my $dir = "$bdir/$sample";
$bamf = $opts{g} if (defined($opts{g}));
$l = $opts{l} if (defined($opts{l}));
$k = $opts{k} if (defined($opts{k}));
$n = $opts{n} if (defined($opts{n}));
$ref = $opts{c} if (defined($opts{c}));
$exec = $opts{e} if (defined($opts{e}));
$no_clipped = 1 if (defined($opts{S}));
$parallel = 1 if (defined($opts{m}));
$clean = 1 if (defined($opts{C}));
$q = $opts{q} if (defined($opts{q}));
$logdir = $opts{L} if (defined($opts{L}));
$step = $opts{a} if (defined($opts{a}));
$nproc = $opts{p} if (defined($opts{p}));
$exogenous = 1 if (defined($opts{x}));
$minram = $opts{M} if (defined $opts{M});
$oneside_ram = 1 if (defined $opts{o});
$family = 1 if (defined $opts{f});
$jittering = $opts{j} if (defined $opts{j});
$skip_cbam = 1 if (defined $opts{F});
$contig = 1 if (defined $opts{t});
$time = $opts{b} if (defined $opts{b});
my $ra = "$tea/lib/assebmly/repeat.combined.div30.isize150.fa";
$ra = $opts{r} if (defined($opts{r}));
$rasym = $opts{R} if (defined($opts{R}));
$scratch = $opts{s} if (defined($opts{s}));
$tea = $opts{P} if (defined($opts{P}));
my $bash_binary = `which bash`;
print "#!$bash_binary\n";
print "export tea_base=$tea\n";
print "export PATH=\$PATH:$tea/R:$tea/scripts\n";
my $ascratch = "$scratch/assembly";
my $sscratch = "$scratch/$sample";
################################################################################
# Note that samtools and tea.pl must be in $PATH for the code below to work.
# Tea requires the below input files in $dir/bam.
# sample.disc.sorted.bam (if exogenous=F)
# sample.cl.disc.sorted.bam (if exogenous=F & no_clipped=F)
# sample.cl.sorted.bam (if exoengeous=T & no_clipped=F)
# sample.mapped_um.sorted.bam (if exoengeous=T & no_clipped=T)
# sample.isinfo
my $bamdir = "$dir/bam";
my $cldir = "$dir/cluster\_${rasym}m";
my $dbamf = "$bamdir/$sample.disc.sorted.bam";
my $dbamf2 = "$bamdir/$sample.disc.bam";
my $clbamf = "$bamdir/$sample.cl.sorted.bam"; # a bam with unmapped and soft-cilpped reads
my $umbamf = "$bamdir/$sample.mapped_um.sorted.bam"; # a bam with unmapped reads
my $umbamf1 = "$bamdir/$sample.um.raw.bam";
my $umbamf2 = "$bamdir/$sample.um.bam";
my $umbz2 = "$bamdir/$sample.umm.bz2";
my $cldbamf = "$bamdir/$sample.cl.disc.sorted.bam";
my $cldbamf2 = "$bamdir/$sample.cl.disc.bam";
my $isf = "$bamdir/$sample.isinfo";
my $isizef = "$bamdir/$sample.isize";
my $rlf = "$bamdir/$sample.rl";
my $cbamf = "$bamdir/$sample.softclips.consd.bam";
my $cbamf2 = "$bamdir/$sample.sorted.softclips.consd.bam";
my $fprefix = "";
if ($step <= 1) {
if ($exogenous && $no_clipped) {
if (!-e $umbamf) {
print STDERR "no bam with unmapped reads: $umbamf";
die "no bam with unmapped reads: $umbamf";
}
else {
print "ln -s $umbamf $umbamf2; ln -s $umbamf.bai $umbamf2.bai\n";
}
} elsif ($exogenous && !$no_clipped) {
if (!-e $clbamf) {
print STDERR "no bam with unmapped and soft clipped reads: $clbamf";
die "no bam with unmapped and soft clipped reads: $clbamf";
} elsif (-e $umbamf2) {
print STDERR "$umbamf2 already exists";
die "$umbamf2 already exists";
} else {
# better make a function for the task below: generating um.bam and umm.bz2
print qq{
echo "generating a bam with unmapped reads ..."
$samtools view -hX $clbamf | perl -ne '\@a=split(/\\t/); if (m/^@/ || \$a[1] =~ /[uU]/) { print } ' | samtools view -bS - > $umbamf1
tea.pl rmdup $umbamf1 $umbamf2
$samtools index $umbamf2
};
print qq{
echo "generating unmapped read positions umm.bz2 ..."
$samtools view -X $umbamf2 | perl -e 'my %h = (); while (<>) { \@a=split(/\\t/); if (\$a[1] =~ m/U/ && !(\$a[1] =~ m/u/) && \$a[4]>0 && (\$_ =~ /XT:A:U/ || !(\$_ =~ /XT:A/))) { \$r = \$a[0]; \$r =~ s/sc\$//; \$r =~ s/mu[12]\$//; if (!exists(\$h{\$r})) { \$a[2] =~ s/chr//; \$h{\$r} = 1; if (\$a[1] =~ /r/) { print "\$a[0]\\t\$a[2]\\t-\$a[3]\\tx\\n"} else { print "\$a[0]\\t\$a[2]\\t\$a[3]\\tx\\n"} } }}' | $bzip2 - > $umbz2
}
}
} else {
if (!-e $dbamf) { die "no $dbamf in $bamdir" }
print "echo \"sorting and generating the index for $dbamf...\"\n";
$fprefix = $dbamf2;
$fprefix =~ s/\.bam$//;
print "$samtools sort -m $sortmem $dbamf $fprefix; $samtools index $fprefix.bam\n";
if (!$no_clipped) {
if (!-e $cldbamf) { die "no $cldbamf in $bamdir" }
print "echo \"sorting and generating the index for $cldbamf...\"\n";
$fprefix = $cldbamf2;
$fprefix =~ s/\.bam$//;
print "$samtools sort -m $sortmem $cldbamf $fprefix; $samtools index $fprefix.bam\n";
}
}
if (!-e $isizef || !-e $rlf) {
if (!-e $isf) { die "$isf is required" }
print "echo \"formatting isize and rl information...\"\n";
print "tea.pl cisize $isf\n";
}
# clean-up
if ($clean) {
print "echo \"removing the input files...\"\n";
print "rm -f $dbamf $clbamf $cldbamf $isf\n";
}
}
# These files should now exist:
# sample.disc.bam (if !exogenous)
# sample.disc.bam.bai (if !exogenous)
# sample.cl.disc.bam (if !exogenous && !no_clipped)
# sample.cl.disc.bam.bai (if !exogenous=F && !no_clipped)
# sample.cl.bam (if exogenous)
# sample.cl.bam.bai (if exogenous)
# sample.isize
# sample.rl
################################################################################
if ($step <=2 && $rasym ne "um") {
if ($exogenous) {
print "echo \"generating fastqs from $umbamf2...\"\n";
print "tea.pl bam2fq -p $umbamf2 $bamdir\n";
} else {
print "echo \"generating fastqs from $dbamf2...\"\n";
print "tea.pl bam2fq -p $dbamf2 $bamdir\n";
if (!$no_clipped) {
print "echo \"generating fastqs from $cldbamf2...\"\n";
print "tea.pl bam2fq -p $cldbamf2 $bamdir\n";
}
}
}
my @fql = ();
if ($exogenous) {
push(@fql, "$sample.um_1.fastq.gz");
push(@fql, "$sample.um_2.fastq.gz");
} else {
push(@fql, "$sample.disc_1.fastq.gz");
push(@fql, "$sample.disc_2.fastq.gz");
if (!$no_clipped) {
push(@fql, "$sample.cl.disc_1.fastq.gz");
push(@fql, "$sample.cl.disc_2.fastq.gz");
}
}
if ($step <=3 && $rasym ne "um") {
if (! -e $ascratch) { print "mkdir -p $ascratch\n"; } # assembly scratch
if (! -e $sscratch) { print "mkdir -p $sscratch\n"; } # sample scratch
if (!-e $ascratch."/".basename($ra).".bwt") {
print "echo \"copying bwa indexes to $ascratch\"\n";
print "cp $ra* $ascratch\n";
}
$ra = $ascratch."/".basename($ra);
for my $fq (@fql) {
my $prefix = $fq;
$prefix =~ s/.fastq.gz//;
$prefix .= ".$rasym";
my $ffq = "$bamdir/$fq";
my $sai = "$sscratch/$prefix.sai";
my $fai = "$ra.fai";
my $rbam = "$bamdir/$prefix.bam";
print qq{
echo "aligning $fq to $ra..."
$bwa aln -t $nproc -l $l -k $k -n $n $ra $ffq > $sai
$bwa samse -n 100 $ra $sai $ffq | $samtools view -bt $fai - > $rbam
};
}
}
if ($step <=4 && $rasym ne "um") {
# generate bams with rams (*.ram.bam) and files with ram positions (*.ram.bz2)\"\n";
my (@rbaml, @rambaml, @rambz2l, $rbam_prefix, $refbam, $rbam1, $rbam2, $rambam, $rambz2);
if ($exogenous) { $rbam_prefix ="$sample.um" }
else { $rbam_prefix ="$sample.disc" }
$refbam = "$rbam_prefix.bam";
$rbam1 = "$rbam_prefix\_1.$rasym.bam";
$rbam2 = "$rbam_prefix\_2.$rasym.bam";
$rambam = "$rbam_prefix.${rasym}m.bam"; # ram or vam
$rambz2 = "$rbam_prefix.${rasym}m.bz2";
print "tea.pl bamram -d $bamdir $refbam $rambam $rambz2 $rbam1 $rbam2\n";
push(@rbaml, "$bamdir/$rbam1"); # for clean-up
push(@rbaml, "$bamdir/$rbam2");
push(@rambaml, "$bamdir/$rambam");
push(@rambz2l, "$bamdir/$rambz2");
if (!$exogenous && !$no_clipped) {
$rbam_prefix ="$sample.cl.disc";
$refbam = "$rbam_prefix.bam";
$rbam1 = "$rbam_prefix\_1.$rasym.bam";
$rbam2 = "$rbam_prefix\_2.$rasym.bam";
$rambam = "$rbam_prefix.${rasym}m.bam";
$rambz2 = "$rbam_prefix.${rasym}m.bz2";
print "tea.pl bamram -d $bamdir $refbam $rambam $rambz2 $rbam1 $rbam2\n";
push(@rbaml, "$bamdir/$rbam1"); # for clean-up
push(@rbaml, "$bamdir/$rbam2");
push(@rambaml, "$bamdir/$rambam");
push(@rambz2l, "$bamdir/$rambz2");
}
# merge ram.bam files and ram.bz2 files
my $temp = "$bamdir/$sample.${rasym}m.raw.bam";
my $m_rambam_prefix = "$bamdir/$sample.${rasym}m";
my $m_rambam = "$m_rambam_prefix.bam";
if (@rambaml >1) {
print "\t\t$samtools merge $temp @rambaml\n";
} else {
print "\t\tmv $rambaml[0] $temp\n";
}
print qq{
$samtools sort -m $sortmem $temp $m_rambam_prefix
$samtools index $m_rambam
rm $temp
};
if (@rambz2l >1) {
print qq{
rm -f $m_rambam_prefix.bz2
bzcat @rambz2l >> $m_rambam_prefix
bzip2 -f $m_rambam_prefix
};
} else {
print "\t\tmv $rambz2l[0] $m_rambam_prefix.bz2\n";
}
# clean-up
if ($clean) { print "rm -f @fql @rbaml @rambaml @rambz2l\n" }
}
# cbam prep
if ($step <=5 && !$skip_cbam ) {
if ($bamf ne "") {
print "tea.pl cbam -o $bamdir $bamf\n";
} elsif (! -e $cbamf) {
die "no $cbamf and no input bam specified to make a clipped bam [-g]";
}
} elsif ($skip_cbam) {
print "echo \"skipping cbam generation\"\n";
}
# run rid
if ($step <=6) {
my @chrl = ();
my %chrl = ();
&get_chrl(\@chrl, \%chrl, $ref);
my $R = "R --no-save --no-restore --no-environ --args $exec";
my $booleanS = "";
# test whether cbam has a 'chr' prefix; for compatibility, check a cbam with 'sorted' infix
my $cbam_chr = 0;
if (-e $cbamf) { $cbam_chr = &bam_chr($cbamf) }
elsif (-e $cbamf2) { $cbam_chr = &bam_chr($cbamf2) }
if ($cbam_chr) {
$booleanS = "cbam.chr=T"
} else {
$booleanS = "cbam.chr=F"
}
if ($no_clipped) { $booleanS .= " no_clipped=T" } else { $booleanS .= " no_clipped=F" }
if ($oneside_ram) { $booleanS .= " oneside.ram=T" } else { $booleanS .= " oneside.ram=F" }
if ($exogenous) { $booleanS .= " exo=T" } else { $booleanS .= " exo=F" }
if ($family) { $booleanS .= " merge.family=T" } else { $booleanS .= " merge.family=F" }
if ($parallel) {
for my $chr (@chrl) {
$chr="chr$chr";
my $clusterf = "$cldir/$sample.$chr.cluster";
my $job = "$sample.$rasym.$chr";
my $logf = "$logdir/$sample.$rasym.log";
if (! -e $clusterf) {
print qq{
echo "running $exec for $chr ..."
bsub -R rusage[mem=10000] -q $q -W $time -g /$sample -J $job -o $logf \\
"$R sample=\\\\\\"$sample\\\\\\\" dir=\\\\\\\"$bdir\\\\\\\" chr=\\\\\\\"$chr\\\\\\\" \\
ref=\\\\\\\"$ref\\\\\\\" rasym=\\\\\\\"$rasym\\\\\\\" min.ram=$minram jittering=$jittering \\
$booleanS < $tea/R/run.rid.r"
};
} else {
print "echo \"skipping $exec for $chr: the cluster file already exists\"\n";
}
}
} else {
print qq{
$R sample=\\"$sample\\" dir=\\"$bdir\\" ref=\\"$ref\\" rasym=\\"$rasym\\" \\
min.ram=$minram jittering=$jittering $booleanS < $tea/R/run.rid.r
};
}
print qq {
echo "pathogen enrichment..."
python $tea/R/pathogen_enrich.py $bamdir/$sample $tea/lib/viruses.txt $dir/cluster_vam/$sample.enrich $dir/cluster_vam/$sample.cluster
}
}
# call germline
if ($step <= 7 && $rasym ne "va") {
my $R = "R --no-save --no-restore --no-environ --args ";
if ($exec eq "rid.te.primate") {
$R .= "te.primate.germline";
} else {
$R .= "germline";
}
my $booleanS;
if ($oneside_ram) { $booleanS .= " oneside.ram=T" } else { $booleanS .= " oneside.ram=F" }
if ($parallel) { $booleanS .= " parallel=T" } else { $booleanS .= " parallel=F" }
if ($contig) { $booleanS .= " contig=T" } else { $booleanS .= " cointig=F" }
my $min_out_conf = 5;
if ($exogenous) { $min_out_conf = 2 };
if ($parallel) {
my $job = "$sample.$rasym.germline";
my $logf = "$logdir/$sample.$rasym.log";
if ($step == 7) {
print qq{
echo "calling germline events ..."
bsub -q short -W 1:00 -g /$sample -J $job \\
-o $logf \\
"$R sample=\\\\\\\"$sample\\\\\\\" dir=\\\\\\\"$bdir\\\\\\\" ref=\\\\\\\"$ref\\\\\\\" \\
rasym=\\\\\\\"$rasym\\\\\\\" min.ram=$minram min.out.conf=$min_out_conf $booleanS < $tea/R/run.rid.r"
}
} else {
print qq{
echo "calling germline events ..."
bsub -q short -W 1:00 -g /$sample -J $job -w $sample.$rasym.chr* \\
-o $logf \\
"$R sample=\\\\\\\"$sample\\\\\\\" dir=\\\\\\\"$bdir\\\\\\\" ref=\\\\\\\"$ref\\\\\\\" \\
rasym=\\\\\\\"$rasym\\\\\\\" min.ram=$minram min.out.conf=$min_out_conf $booleanS < $tea/R/run.rid.r"
}
}
} else {
print qq{
$R sample=\\"$sample\\" dir=\\"$bdir\\" ref=\\"$ref\\" rasym=\\"$rasym\\" \\
min.ram=$minram min.out.conf=$min_out_conf $booleanS < $tea/R/run.rid.r
};
}
}
print "echo \"######## complete ##########\"\n";
}
sub bam_chr
{
my $bamf = @_[0];
my @headers = `samtools view -H $bamf`;
my $bam_chr = 0;
for my $h (@headers) {
if ($h =~ m/^\@SQ/) {
$h =~ s/.*SN:(.*)\s.*/$1/;
if ($h =~ m/chr/) {
$bam_chr = 1;
last;
}
}
}
return $bam_chr;
}
sub rmasker_coordinates
{
my ($rmasker_file, $rmasker_start_line, $verbose) = @ARGV;
my $outf = $rmasker_file;
$outf =~ s/gz/coordinates.gz/;
open(OUT, "| gzip -c > $outf") || die "can't create $outf: $!";
# read rmasker and get the instance coordinates
print "reading repeatmasker file $rmasker_file ...\n" if ($verbose);
open(RM, "zcat $rmasker_file | ") || die "can't open repeat masker file $rmasker_file: $!";
my $cnt=1;
while ( $cnt++ < $rmasker_start_line) { <RM> }
my $icnt = 0;
my %reppos;
my ($name, $divscore, $chr, $s, $e);
while (<RM>) {
chomp; my $line=$_;
$line=~s/^\s+//;
my @data = split(/\s+/,$line);
#$name = "$data[9]:$data[10]:$data[11]";
$data[10] =~ s/_$//;# remove the tailing _
$name = "$data[10]:$data[11]:$data[12]";
$divscore = $data[2]+$data[3]+$data[4];
$chr = $data[5];
$s = $data[6]+1; # note that all rmasker start coordinates are 0-t()based not 1-based
$e = $data[7];
push @{$reppos{uc($name)}}, "$chr:$s-$e";
#push @{$reppos{$name}}, "$chr:$s-$e";
$icnt++;
}
for my $n (keys %reppos) {
print OUT "$n\t@{$reppos{$n}}\n";
}
close OUT;
print "done extracting $icnt repeat instance coordinates of ".scalar(keys %reppos)." repeat types.\n" if ($verbose);
}
sub rmasker_coordinates_strand
{
my ($rmasker_file, $rmasker_start_line, $verbose) = @ARGV;
my $outf = $rmasker_file;
$outf =~ s/gz/coordinates.strand.gz/;
open(OUT, "| gzip -c > $outf") || die "can't create $outf: $!";
# read rmasker and get the instance coordinates
print "reading repeatmasker file $rmasker_file ...\n" if ($verbose);
open(RM, "zcat $rmasker_file | ") || die "can't open repeat masker file $rmasker_file: $!";
my $cnt=1;
while ( $cnt++ < $rmasker_start_line) { <RM> }
my $icnt = 0;
my %reppos;
my ($name, $divscore, $chr, $s, $e, $strand, $div);
while (<RM>) {
chomp; my $line=$_;
$line=~s/^\s+//;
my @data = split(/\s+/,$line);
#$name = "$data[9]:$data[10]:$data[11]";
$data[10] =~ s/_$//;# remove the tailing _
$name = "$data[10]:$data[11]:$data[12]";
$divscore = $data[2]+$data[3]+$data[4];
$chr = $data[5];
$s = $data[6]+1; # note that all rmasker start coordinates are 0-t()based not 1-based
$e = $data[7];
$strand = $data[9];
push @{$reppos{uc($name)}}, "$chr:$s,$e:$strand:$divscore";
#push @{$reppos{$name}}, "$chr:$s-$e";
$icnt++;
}
for my $n (keys %reppos) {
print OUT "$n\t@{$reppos{$n}}\n";
}
close OUT;
print "done extracting $icnt repeat instance coordinates of ".scalar(keys %reppos)." repeat types.\n" if ($verbose);
}
sub make_subfamilyseq
{
my ($family, $map_subfamily, $rdbs) = @_;
my ($cmd, $db, $diag);
if ($family eq "L1") {
$db = "/home/el114/repeat_analysis/data/consensus/M80343.1.fasta";
} elsif (uc($family) eq "ALU") {
$db = "/home/el114/repeat_analysis/data/consensus/AluY.fasta";
} elsif ($family eq "PABL_A-INT") {
$db = "/home/el114/repeat_analysis/data/consensus/PABL_A-INT.fasta";
}
push(@$rdbs, $db);
if (!$map_subfamily) { return; }
if ($family eq "L1") {
open(L, "< $db") || die "can't open $db";
my $seq = <L>; $seq = ""; while (<L>) { chomp; $seq = $seq.$_; } # get the consensus seq
# print length($seq)."\n";
$diag = "/home/el114/repeat_analysis/data/consensus/diagnostic_L1_positions.txt";
open(D, "< $diag") || die "can't open $diag\n";
my @m = <D>; chomp(@m);
my @subfamily = split(/\t/, $m[0]); shift(@subfamily);
my %seqs = (); foreach (@subfamily) { $seqs{$_} = $seq; } # subfamily consensus seq initialization
my (@a, $pos);
for (my $i=1; $i<@m; $i++) {
@a = split(/\t/, $m[$i]);
$pos = shift(@a);
for (my $j=0; $j<@a; $j++) { if ($a[$j] ne "-") {
#print "replacing $subfamily[$j] $pos, to $a[$j]\n";
substr($seqs{$subfamily[$j]}, $pos-1, 1, uc($a[$j])); }
}
}
foreach (@subfamily) {
my $fasta = $db;
$fasta =~ s/(.*).fasta/$1/;
$fasta = "${fasta}_$_.fasta";
push(@$rdbs, $fasta);
open(O, "> $fasta") || die "can't create $fasta";
print O ">$_\n$seqs{$_}\n";
$cmd = "$blast_path/makeblastdb -dbtype nucl -in $fasta";
print "generating blast db for $fasta..\n";
system($cmd);
print "done generating.\n";
}
} elsif (uc($family) eq "Alu") {
push(@$rdbs, "/home/el114/repeat_analysis/data/consensus/AluYa5.fasta");
push(@$rdbs, "/home/el114/repeat_analysis/data/consensus/AluYb8.fasta");
push(@$rdbs, "/home/el114/repeat_analysis/data/consensus/AluYb9.fasta");
push(@$rdbs, "/home/el114/repeat_analysis/data/consensus/AluSc.fasta");
}
}
# blast clipped and ram mate contigs to consensus sequences
# and annotate insertion size, inversion status and diagnostic features for L1
# requires unique ids, otherwise generates ids [family_##]
sub map_consensus
{
my ($file, $family, $map_subfamily, $verbose) = @_;
open(F, "< $file") || die "can'e open $file";
open(O, "> $file.fasta") || die "can't create $file.fasta"; # fasta
open(O2, "> $file.annot") || die "can't create $file.annot"; # full annot
my $header = <F>; chomp($header);
my @a = split(/\t/, $header); my %idx = ();
for (my $i=0; $i<@a; $i++) { $idx{$a[$i]} = $i; }
my (%insertions, $sname, $seq); # id to record
print "reading $file and generating $file.fasta..\n";
my @f = <F>;
my @ids = ();
if (exists $idx{id}) {
for (my $i=0; $i<@f; $i++) {
chomp($f[$i]); my @a=split(/\t/, $f[$i]);
if ($a[$idx{family}] eq $family) {
push(@ids, $a[$idx{id}]);
$insertions{$a[$idx{id}]}{line} = $f[$i];
$insertions{$a[$idx{id}]}{orientation} = $a[$idx{orientation}];
$insertions{$a[$idx{id}]}{polyA} = $a[$idx{polyA}];
$insertions{$a[$idx{id}]}{polyT} = $a[$idx{polyT}];
$sname = "$a[$idx{id}]:pclipped";
$seq = $a[$idx{pclipped}];
print O ">$sname\n$seq\n";
$sname = "$a[$idx{id}]:nclipped";
$seq = $a[$idx{nclipped}];
print O ">$sname\n$seq\n";
$sname = "$a[$idx{id}]:prammate";
$seq = $a[$idx{prammate}];
print O ">$sname\n$seq\n";
$sname = "$a[$idx{id}]:nrammate";
$seq = $a[$idx{nrammate}];
print O ">$sname\n$seq\n";
}
}
} else { # generate unique id
my ($cnt, $uid) = (1, "");
for (my $i=0; $i<@f; $i++) {
chomp($f[$i]); my @a=split(/\t/, $f[$i]);
# for the modification for the young repeat families (L1HS) and polyA support
#if ($a[$idx{family}] eq $family) {
if ($a[$idx{family}] =~ m/$family/ || $a[$idx{"rep.repeat"}] =~ m/$family/) {
$uid = "$family\_$cnt"; $cnt++;
push(@ids, $uid);
$insertions{$uid}{line} = "$uid\t$f[$i]";
$insertions{$uid}{orientation} = $a[$idx{orientation}];
$insertions{$uid}{polyA} = $a[$idx{polyA}];
$insertions{$uid}{polyT} = $a[$idx{polyT}];
$sname = "$uid:pclipped";
$seq = $a[$idx{pclipped}];
print O ">$sname\n$seq\n";
$sname = "$uid:nclipped";
$seq = $a[$idx{nclipped}];
print O ">$sname\n$seq\n";
$sname = "$uid:prammate";
$seq = $a[$idx{prammate}];
print O ">$sname\n$seq\n";
}
}
$header = "id\t$header";
}
print "done generating.\n";
close F; close O;
my @dbs = (); # db for each subfamily
&make_subfamilyseq($family, $map_subfamily, \@dbs);
my @asf = ();
for my $sf (@dbs) {
my $subfamily = basename($sf); $subfamily =~ s/(.*).fasta/$1/;
push(@asf, $subfamily);
print "subfamily: $subfamily\n";
print "mapping consensus for $subfamily\n";
&map_consensus_db(\%insertions, "$file.fasta", $sf, $subfamily, $verbose); # inversion, insertion size, %divergence
}
print O2 "$header\tcontig_orientation\torientation\tinversion\tsize\tcstart\tcend\tsubfamily\tmismatch\tdivpct\n";
for my $id (@ids) {
print O2 "$insertions{$id}{line}";
# summarize divs and extract subfamilies with the minimum div
my (@divs, @divpcts, @est_subfamily);
my $mindiv = 9999;
for my $sf (@asf) {
my $ssf = $sf; # short sf removing M80343.1 prefix
#if (($family eq "L1" || $family eq "L1HS" ) && $sf ne "M80343.1") { # skip L1 ref name
if ($family =~ m/L1/ && $sf ne "M80343.1") { # skip L1 ref name
$ssf =~ s/M80343.1_//;
}
push(@divs, "$ssf:$insertions{$id}{$sf}{div}");
push(@divpcts, "$ssf:$insertions{$id}{$sf}{divpct}");
if ($insertions{$id}{$sf}{div} < $mindiv) { $mindiv = $insertions{$id}{$sf}{div} }
}
for my $sf (@asf) {
if ($sf eq "M80343.1") { next }
my $ssf = $sf; $ssf =~ s/M80343.1_//;
if ($insertions{$id}{$sf}{div} == $mindiv) { push(@est_subfamily, $ssf) }
}
if (scalar @est_subfamily == (scalar @divs)-1) { @est_subfamily = (); }
# decide the final orientation from polyA/T and contig mapping
my $orientation = $insertions{$id}{orientation};
if ($orientation eq "NA" && exists $insertions{$id}{$asf[0]}{map_orientation}) {
$orientation = $insertions{$id}{$asf[0]}{map_orientation};
}
if (exists $insertions{$id}{$asf[0]}{map_orientation}) {
print O2 "\t$insertions{$id}{$asf[0]}{map_orientation}\t$orientation\t$insertions{$id}{$asf[0]}{inv}\t$insertions{$id}{$asf[0]}{size}\t$insertions{$id}{$asf[0]}{cstart}\t$insertions{$id}{$asf[0]}{cend}\t";
if (@est_subfamily >=1) {
print O2 join(",", @est_subfamily)."\t";
} else {
print O2 "-\t";
}
print O2 join(",", @divs)."\t";
print O2 join(",", @divpcts)."\n";
} else {
print O2 "\tNA\tNA\tNA\tNA\tNA\tNA\tNA\tNA\tNA\n";
}
}
close O2;
`rm $file.fasta`;
if (!$verbose) { `rm $file.*.fasta.map` }
}
sub map_consensus_db
{
my ($href, $fasta, $db, $subfamily, $verbose) = @_;
print "blasting $fasta to $db..\n";
my $cmd = "$blast_path/legacy_blast.pl blastall -p blastn -m 8 -i $fasta -d $db --path $blast_path > $fasta.".basename($db).".map";
print $cmd."\n"; system($cmd);
print "done.\n";
open(F, "< $db") || die "can't open $db";
my $l = <F>; $l = ""; while (<F>) { chomp; $l = $l.$_; } $l = length($l);
&parse_map($href, $fasta.".".basename($db).".map", $l, $subfamily, $verbose);
if (!$verbose) {
$cmd = "rm $fasta.".basename($db).".map";
#system($cmd);
}
}
sub parse_map
{
my ($href, $mapfile, $full_length, $sf, $verbose) = @_;
# fille the hash for each id; if two mappings then chose the longer mapping
open(F, "< $mapfile") || die "can't open $mapfile";
print "reading $mapfile\n";
while (<F>) {
chomp;
my ($qid, $sid, $identity, $alnlen, $mismatch, $gap, $qstart, $qend, $sstart, $send, $evalue, $bitscore) = split(/\t/);
#print "$qid, $sid, $identity, $alnlen, $mismatch, $gap, $qstart, $qend, $sstart, $send, $evalue, $bitscore\n";
my ($id, $type) = ($qid, $qid); $id =~ s/(.*):.*/$1/; $type =~ s/.*:(.*)/$1/;
if (!exists$href->{$id}{$sf}{$type} || ($alnlen > $href->{$id}{$sf}{$type}{alnlen}) || ($$alnlen == $href->{$id}{$sf}{$type}{alnlen} && $href->{$id}{$sf}{$type}{mismatch} > $mismatch)) { # choose the maximum match and minimum mismaches where thee are multiple mappings
$href->{$id}{$sf}{$type}{alnlen} = $alnlen;
$href->{$id}{$sf}{$type}{mismatch} = $mismatch;
$href->{$id}{$sf}{$type}{gap} = $gap;
$href->{$id}{$sf}{$type}{sstart} = $sstart;
$href->{$id}{$sf}{$type}{send} = $send;
if ($sstart <= $send) {
$href->{$id}{$sf}{$type}{orientation} = "+";
} else {
$href->{$id}{$sf}{$type}{orientation} = "-";
}
}
}
for my $id (keys %{$href}) {
my ($orientation, $inv, $start, $end, $size, $alnlen, $div, $divpct) = ("NA", "NA", -1, -1, -1, 0, 0, -1);
if (exists $href->{$id}{$sf}) {
for my $type (keys %{$href->{$id}{$sf}}) {
for my $key (keys %{$href->{$id}{$sf}{$type}}) {
if ($verbose) { print "$id->$sf->$type->$key: $href->{$id}{$sf}{$type}{$key}\n"; }
}
$div = $div + $href->{$id}{$sf}{$type}{mismatch} + $href->{$id}{$sf}{$type}{gap};
$alnlen = $alnlen + $href->{$id}{$sf}{$type}{alnlen};
}
# orientation: + or - if both prammate (nclipped) and nrammate (pclipped) are + or -
my ($l, $r) = ("NA", "NA");
if (exists $href->{$id}{$sf}{prammate}{orientation}) {
$l = $href->{$id}{$sf}{prammate}{orientation};
} elsif (exists $href->{$id}{$sf}{nclipped}{orientation}) {
$l = $href->{$id}{$sf}{nclipped}{orientation};
}
if (exists $href->{$id}{$sf}{nrammate}{orientation}) {
$r = $href->{$id}{$sf}{nrammate}{orientation};
} elsif (exists $href->{$id}{$sf}{pclipped}{orientation}) {
$r = $href->{$id}{$sf}{pclipped}{orientation};
}
if ($l ne "NA" && $r ne "NA") {
if ($l eq "+" && $r eq "+") {
$orientation = "+";
$start = 9999;
if (exists $href->{$id}{$sf}{prammate}{sstart} && $href->{$id}{$sf}{prammate}{sstart}< $start) { $start = $href->{$id}{$sf}{prammate}{sstart}}
if (exists $href->{$id}{$sf}{nclipped}{sstart} && $href->{$id}{$sf}{nclipped}{sstart}< $start) { $start = $href->{$id}{$sf}{nclipped}{sstart}}
if (exists $href->{$id}{$sf}{nrammate}{sstart} && $href->{$id}{$sf}{nrammate}{sstart}< $start) { $start = $href->{$id}{$sf}{nrammate}{sstart}}
$end = -1;
if (exists $href->{$id}{$sf}{nrammate}{send} && $href->{$id}{$sf}{nrammate}{send} > $end) { $end = $href->{$id}{$sf}{nrammate}{send}}
if (exists $href->{$id}{$sf}{pclipped}{send} && $href->{$id}{$sf}{pclipped}{send} > $end) { $end = $href->{$id}{$sf}{pclipped}{send}}
if (exists $href->{$id}{$sf}{prammate}{send} && $href->{$id}{$sf}{prammate}{send} > $end) { $end = $href->{$id}{$sf}{prammate}{send}}
$size = $end - $start + 1;
$inv = "NO_INV";
} elsif ($l eq "-" && $r eq "-") {
$orientation = "-";
$start = 9999;
if (exists $href->{$id}{$sf}{nrammate}{send} && $href->{$id}{$sf}{nrammate}{send}< $start) { $start = $href->{$id}{$sf}{nrammate}{send}}
if (exists $href->{$id}{$sf}{pclipped}{send} && $href->{$id}{$sf}{pclipped}{send}< $start) { $start = $href->{$id}{$sf}{pclipped}{send}}
if (exists $href->{$id}{$sf}{prammate}{send} && $href->{$id}{$sf}{prammate}{send}< $start) { $start = $href->{$id}{$sf}{prammate}{send}}
$end = -1;
if (exists $href->{$id}{$sf}{prammate}{sstart} && $href->{$id}{$sf}{prammate}{sstart} > $end) { $end = $href->{$id}{$sf}{prammate}{sstart}}
if (exists $href->{$id}{$sf}{nclipped}{sstart} && $href->{$id}{$sf}{nclipped}{sstart} > $end) { $end = $href->{$id}{$sf}{nclipped}{sstart}}
if (exists $href->{$id}{$sf}{nrammate}{sstart} && $href->{$id}{$sf}{nrammate}{sstart} > $end) { $end = $href->{$id}{$sf}{nrammate}{sstart}}
$inv = "NO_INV";
} else {
$inv = "INV";
}
} elsif ($l eq "NA" && $href->{$id}{polyT} eq "polyT") {
$orientation = "-";
if ($r eq "-") {
$inv = "NO_INV";
$start = 9999;
if (exists $href->{$id}{$sf}{nrammate}{send} && $href->{$id}{$sf}{nrammate}{send}< $start) { $start = $href->{$id}{$sf}{nrammate}{send}}
if (exists $href->{$id}{$sf}{pclipped}{send} && $href->{$id}{$sf}{pclipped}{send}< $start) { $start = $href->{$id}{$sf}{pclipped}{send}}
if (exists $href->{$id}{$sf}{prammate}{send} && $href->{$id}{$sf}{prammate}{send}< $start) { $start = $href->{$id}{$sf}{prammate}{send}}
$end = $full_length;
} else {
$inv = "INV";
}
} elsif ($r eq "NA" && $href->{$id}{polyA} eq "polyA") {
$orientation = "+";
if ($l eq "+") {
$inv = "NO_INV";
$start = 9999;
if (exists $href->{$id}{$sf}{prammate}{sstart} && $href->{$id}{$sf}{prammate}{sstart}< $start) { $start = $href->{$id}{$sf}{prammate}{sstart}}
if (exists $href->{$id}{$sf}{nclipped}{sstart} && $href->{$id}{$sf}{nclipped}{sstart}< $start) { $start = $href->{$id}{$sf}{nclipped}{sstart}}
if (exists $href->{$id}{$sf}{nrammate}{sstart} && $href->{$id}{$sf}{nrammate}{sstart}< $start) { $start = $href->{$id}{$sf}{nrammate}{sstart}}
$end = $full_length;
} else {
$inv = "INV";
}
}
if ($start != -1 && $end != -1) { $size = $end - $start + 1; }
$divpct = sprintf("%.1f", $div/$alnlen*100);
$href->{$id}{$sf}{map_orientation} = $orientation;
$href->{$id}{$sf}{inv} = $inv;
$href->{$id}{$sf}{cstart} = $start;
$href->{$id}{$sf}{cend} = $end;
$href->{$id}{$sf}{size} = $size;
$href->{$id}{$sf}{divpct} = $divpct;
$href->{$id}{$sf}{div} = $div;
print "$l: $r: $id: $sf: $orientation, $inv, $size, $start, $end, $div, $divpct, $alnlen\n";
}
}
}
sub contig {
my $usage = qq {
Usage: contig [options] <input file>
Options: -a STR path to the contig assembler (cap3)
-d STR path to RUN DIRECTORY (upper to the sample folder)
with read names and clipped sequences and disc. bam files)
-o STR outdir (.)
-C INT confidence level for filtering (0)
-m STR consensus mapping [L1|Alu|SVA] (default: no)
-s subfamily mapping (default: no)
-t STR tempdir for running cap3 (/hms/scratch1/el114)
-c clipped sequence contig only (0)
-r ram mate contig only (0)
-e extended ram mate contig only (0)
-x exogenous (0)
-p Tea was run for each chromosome (0)
-R STR assembly symbol
-v verbose
};
my %opts = ();
getopts("ha:d:m:o:vrcevst:C:xR:p", \%opts);
if (@ARGV < 1 || exists($opts{h})) { die $usage };
my $verbose = 0; $verbose = 1 if (defined $opts{v});
my $conly = 0; $conly = 1 if (defined $opts{c});
my $ronly = 0; $ronly = 1 if (defined $opts{r});