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chk_feature2count.pl
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chk_feature2count.pl
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#!/usr/bin/env perl
##################################
# count reads on each input seq
# using HTSeq-count
##################################
use strict;
use warnings;
use Cwd qw(abs_path cwd);
use File::Which;
use File::Path qw(make_path remove_tree);
use File::Basename qw(dirname basename);
use File::Spec::Functions qw(catdir catfile);
use Getopt::Std;
use Data::Dumper;
use FindBin qw($Bin);
use lib catdir(dirname($Bin), 'lib');
my %func = ('samtools' => '',
'bedtools' => '',
'htseq-count' => '',
'sort2bed.pl' => '',
'featureCounts' => '');
my $f = check_tools(\%func);
%func = %{$f};
&seq2count();
#exit(1);
sub seq2count {
my %opts = (o => 'Count_seqs', p => 'featureCounts', s => 'yes');
getopts('o:l:f:s:p:', \%opts);
&usage if(@ARGV == 0);
die("[-l] Need bam list\n") if(! defined($opts{l}));
die("[-f] Need reference *.fa n") if(! defined($opts{f}));
die("[-p: $opts{p}] can be: htseq-count or bedtools.") if(! $opts{p} =~ /^(htseq-count)|(bedtools)|(featureCounts)$/);
die("[-s: $opts{s}] can be: yes, no or reverse") if(! $opts{s} =~ /^(yes)|(reverse)$/);
$opts{o} = abs_path($opts{o});
$opts{f} = abs_path($opts{f});
make_path($opts{o}) if(! -d $opts{o});
my $infile = abs_path( shift(@ARGV) );
my @bams = readBAMlist($opts{l});
my $ref = $opts{f};
my @runs = ();
my $num = 1;
my $out_count = catfile($opts{o}, basename($infile));
$out_count =~ s/\.txt/_count.txt/;
# create temp dir
my $origin_path = abs_path(cwd);
my $temp_dir = catdir(cwd, 'temp'.'.'.$$.'_'.int(rand(1000000)));
make_path($temp_dir) if(! -d $temp_dir);
chdir $temp_dir;
for my $b (sort @bams) { my $flag = sprintf"%0d", $num; if($opts{p} eq 'htseq-count' ) {
push @runs, htseq_count($b, $infile, $flag, $opts{s}, $temp_dir);
}elsif($opts{p} eq 'bedtools') {
push @runs, bedtools_count($b, $infile, $flag, $opts{s}, $temp_dir);
}elsif($opts{p} eq 'featureCounts') {
push @runs, featureCounts_count($b, $infile, $flag, $opts{s}, $temp_dir);
}
$num ++;
}
if($opts{p} eq 'featureCounts') {
my $tmp_summary = catfile($temp_dir, basename($infile) . '.tmp.summary.*');
my $out_summary = $out_count;
$out_summary =~ s/\.txt/.summary/;
push @runs, "cat $tmp_summary > $out_summary";
}
my $tmp2_file = catfile($temp_dir, basename($infile).'.tmp2');
my $TPM_files = catfile($temp_dir, basename($infile).'.TPM.*');
push @runs, "sort -k1 $infile -o $infile";
push @runs, "paste $infile $TPM_files > $out_count";
for my $r (@runs) {
print $r . "\n";
system"$r";
}
chdir $origin_path;
remove_tree($temp_dir);
}
#sub count_seq {
sub htseq_count {
my ($bam, $in, $num, $strand, $outdir) = @_;
# determine strand
my $bam_name = basename($bam);
$bam_name =~ s/(\.|\.s.|\.f.s.|\.trim\.gz\.f\.s\.)bam//;
my $lib_type = '';
if($strand eq 'yes') {
$lib_type = ($bam_name =~ /\_[12]$/)?'reverse':'yes'; # PE=reverse, SE=yes
}elsif($strand eq 'reverse') {
$lib_type = ($bam_name =~ /\_[12]$/)?'yes':'reverse';
}else {
#
}
# run
my @runs = ();
chomp(my $chr = qx($func{'samtools'} view $bam | head -n1 | awk '{print \$3}'));
### fix bug: chr name with '|'
$chr =~ s/\|/\\\|/g;
###
my $in_gff = basename($in);
$in_gff =~ s/\.txt$/.gff/;
$in_gff = catfile($outdir, $in_gff);
my $in_tmp = catfile($outdir, basename($in).'.tmp');
my $in_tmp2 = catfile($outdir, basename($in).'.tmp2');
my $in_TPM = catfile($outdir, basename($in).'.TPM.'.$num);
if( not_blank_file($in) ) {
push @runs, "perl $func{'sort2bed.pl'} -t sort2gff -f exon -s $chr -i $in -o $in_gff";
push @runs, "$func{'htseq-count'} -q -f bam -s $lib_type -t exon $bam $in_gff > $in_tmp";
push @runs, "sort -k1 $in_tmp | sed -e \'/^\_/d\' > $in_tmp2";
# count tpm
my $bam_mapped = qx($func{'samtools'} idxstats $bam | head -n1 |awk '{print \$3}');
my $ratio = sprintf"%.4f", 1000000/$bam_mapped;
push @runs, "awk \'{printf(\"\%s\\t\%s\\t\%.4f\\n\", \$1, \$2, \$2*$ratio)}\' \< $in_tmp2 | cut -f2-3 > $in_TPM";
}
return @runs;
}
sub featureCounts_count {
my ($bam, $in, $num, $strand, $outdir) = @_;
# determine strand
my $bam_name = basename($bam);
$bam_name =~ s/(\.|\.s.|\.f.s.|\.trim\.gz\.f\.s\.)bam//;
my $lib_type = '';
if($strand eq 'yes') {
$lib_type = ($bam_name =~ /\_[12]$/)?'2':'1'; # PE=2, SE=1
}elsif($strand eq 'reverse') {
$lib_type = ($bam_name =~ /\_[12]$/)?'1':'2'; # reverse
}else {
#
}
my @runs = ();
chomp(my $chr = qx($func{'samtools'} view $bam | head -n1 | awk '{print \$3}'));
### fix bug: chr name with '|'
$chr =~ s/\|/\\\|/g;
###
my $in_gff = basename($in);
$in_gff =~ s/\.txt$/.gff/;
$in_gff = catfile($outdir, $in_gff);
my $in_tmp = catfile($outdir, basename($in).'.tmp');
my $in_tmp2 = catfile($outdir, basename($in).'.tmp2');
my $in_TPM = catfile($outdir, basename($in).'.TPM.'.$num);
my $fc_para = '';
if($bam_name =~ /\_[1-9]+$/) { # PE reads
$fc_para = join(" ", '-M --fraction --donotsort -O -f -T 5 -g gene_id -t exon -p -P -d 40 -D 500 -s', $lib_type, '-a', $in_gff, '-o', $in_tmp);
# $fc_para = join(" ", '-M --fraction --donotsort -f -T 5 -g gene_id -t exon -p -P -d 40 -D 500 -s', $lib_type, '-a', $in_gff, '-o', $in_tmp);
}else {
$fc_para = join(" ", '-M --fraction --donotsort -O -f -T 5 -g gene_id -t exon -s', $lib_type, '-a', $in_gff, '-o', $in_tmp);
# $fc_para = join(" ", '-M --fraction --donotsort -f -T 5 -g gene_id -t exon -s', $lib_type, '-a', $in_gff, '-o', $in_tmp);
}
if( not_blank_file($in) ) {
push @runs, "perl $func{'sort2bed.pl'} -t sort2gff -f exon -s $chr -i $in -o $in_gff";
push @runs, "$func{'featureCounts'} $fc_para $bam > $in\.log 2>&1";
# push @runs, "$func{'featureCounts'} -p -P -d 40 -D 500 -C -T 5 -s $lib_type -M --fraction -O -f --donotsort -t exon -g gene_id -a $in_gff -o $in_tmp $bam >>$in\.log 2>&1"; # need: -O
# keep summary
push @runs, "mv $in_tmp\.summary $in_tmp\.summary\.$num";
push @runs, "cat $in_tmp | sed \'1,2 d\' | sort -k1 > $in_tmp2";
# count tpm
my $bam_mapped = qx($func{'samtools'} idxstats $bam | head -n1 |awk '{print \$3}');
my $ratio = sprintf"%.2f", 1000000/$bam_mapped;
push @runs, "awk \'{printf(\"\%s\\t\%s\\t\%.4f\\n\", \$1, \$7, \$7*$ratio)}\' \< $in_tmp2 | cut -f2-3 > $in_TPM";
}
return @runs;
}
sub bedtools_count {
my ($bam, $in, $num, $strand, $outdir) = @_;
my $bam_name = basename($bam);
$bam_name =~ s/(\.|\.s.|\.f.s.|\.trim\.gz\.f\.s\.)bam//;
my @runs = ();
my $str_type = '';
if($strand eq 'yes') {
$str_type = '-s';
}elsif($strand eq 'reverse') {
$str_type = '-S';
}else {
$str_type = '';
}
my $chr = qx($func{'samtools'} view $bam | head -n1 | awk '{print \$3}');
chomp($chr);
### fix bug: chr name with '|'
$chr =~ s/\|/\\\|/g;
###
my $in_bed = basename($in);
$in_bed =~ s/\.txt/.bed/;
$in_bed = catfile($outdir, $in_bed);
my $in_tmp = catfile($outdir, basename($in).'.tmp');
my $in_TPM = catfile($outdir, basename($in).'.TPM.'.$num);
if( not_blank_file($in) ) {
push @runs, "perl $func{'sort2bed.pl'} -t sort2bed -s $chr -i $in -o $in_bed";
# push @runs, "$func{'bedtools'} intersect -c $str_type -a $in_bed -b $bam > $in_tmp";
push @runs, "$func{'bedtools'} multicov -split -D $str_type -bams $bam -bed $in_bed > $in_tmp";
# count tpm
my $bam_mapped = qx($func{'samtools'} idxstats $bam | head -n1 |awk '{print \$3}');
my $ratio = sprintf"%.2f", 1000000/$bam_mapped;
push @runs, "awk \'{printf(\"\%s\\t\%s\\t\%.4f\\n\", \$1, \$7, \$7*$ratio)}\' \< $in_tmp | cut -f2-3 > $in_TPM";
}
return @runs;
}
sub readBAMlist {
my $in = shift(@_);
die("[$in] BAM list file not found.\n") if(! -e $in);
my @bams = ();
open my $fh_in, "< $in" or die "$!";
while(<$fh_in>) {
chomp;
next if(/(^\s*$)|(^\#)/);
my $b = (split /\s+/)[0];
die("[$b] in line:$. of $in, not found\n") if(! -e $b);
push @bams, abs_path($b);
}
close $fh_in;
return @bams;
}
sub not_blank_file {
my $in = shift(@_);
if( -e $in) {
open my $fh_in, "< $in" or die "$!";
my $num = 0;
while(<$fh_in>) {
chomp;
next if(/(^\s*$)|(^\#)/);
$num ++;
}
close $fh_in;
return $num;
}else {
return 0;
}
}
# htseq-count
# samtools
sub check_tools {
my $tool = shift(@_);
my %func = %{$tool};
my @missing = ();
for my $f (sort keys %func) {
if( tool_path($f) ) {
$func{$f} = tool_path($f);
}else {
push @missing, $f;
}
}
if(@missing) {
print 'The following tool(s) missing:' . "\n\n";
my $err_log = '';
for my $m (sort @missing) {
$err_log .= sprintf("%-15s : Not found in \$PATH, \$Bin, or ~/work/bin/temp/\n", $m);
}
die("$err_log\n");
}
my $flag = (@missing)?0:1;
return($flag, \%func);
#
sub tool_path {
my $t = shift(@_);
my $perl_path = $ENV{HOME}. '/work/bin/temp';
if( -e catfile($perl_path, $t)) {
return catfile($perl_path, $t);
}elsif( which($t) ) {
return $t;
}elsif( -e catfile($Bin, $t) ) {
return catfile($Bin, $t);
}else {
return 0;
}
}
}
sub usage{
die(qq/
Usage: chk_seq2count.pl [options] <in.txt>
Options: -o Output path. [Count_seqs]
-l The bam list files, only one bam on each line
-f The reference Fasta file
-s Strand : yes no reverse [yes]
-p The program to count reads: bedtools, htseq-count, featureCounts
[featureCounts]
\n/);
}
__END__
Changelog
2015-08-08
1.change TPM to 2-digits format