-#!/usr/bin/perl
+#!/usr/bin/env perl
use Getopt::Long;
use Pod::Usage;
-use FindBin;
-use lib $FindBin::Bin;
-use strict;
+use FindBin;
+use lib $FindBin::RealBin;
use rsem_perl_utils qw(runCommand collectResults showVersionInfo);
+use Env qw(@PATH);
+@PATH = ($FindBin::RealBin, "$FindBin::RealBin/sam", @PATH);
+
+use strict;
+
#const
my $BURNIN = 200;
my $NCV = 1000;
my $NSPC = 50;
my $NMB = 1024; # default
+my $SortMem = "1G"; # default as 1G per thread
my $status = 0;
my $genBamF = 1; # default is generating transcript bam file
my $genGenomeBamF = 0;
my $sampling = 0;
+my $calcPME = 0;
my $calcCI = 0;
-my $var_opt = 0; # temporarily, only for internal use
my $quiet = 0;
my $help = 0;
my $bowtie2_k = 200;
my $bowtie2_sensitivity_level = "sensitive"; # must be one of "very_fast", "fast", "sensitive", "very_sensitive"
+my $seed = "NULL";
+
my $version = 0;
my $mTime = 0;
my ($refName, $sampleName, $sampleToken, $temp_dir, $stat_dir, $imdName, $statName) = ();
my $gap = 32;
+my $alleleS = 0;
+
GetOptions("keep-intermediate-files" => \$keep_intermediate_files,
"temporary-folder=s" => \$temp_dir,
"no-qualities" => \$no_qual,
"no-bam-output" => sub { $genBamF = 0; },
"output-genome-bam" => \$genGenomeBamF,
"sampling-for-bam" => \$sampling,
- "var" => \$var_opt,
+ "calc-pme" => \$calcPME,
"calc-ci" => \$calcCI,
"ci-memory=i" => \$NMB,
+ "samtools-sort-mem=s" => \$SortMem,
+ "seed=i" => \$seed,
"time" => \$mTime,
"version" => \$version,
"q|quiet" => \$quiet,
"h|help" => \$help) or pod2usage(-exitval => 2, -verbose => 2);
-my $dir = "$FindBin::Bin/";
-
pod2usage(-verbose => 2) if ($help == 1);
-&showVersionInfo($dir) if ($version == 1);
+&showVersionInfo($FindBin::RealBin) if ($version == 1);
#check parameters and options
pod2usage(-msg => "Seed length should be at least 5!\n", -exitval => 2, -verbose => 2) if ($L < 5);
pod2usage(-msg => "--sampling-for-bam cannot be specified if --no-bam-output is specified!\n", -exitval => 2, -verbose => 2) if ($sampling && !$genBamF);
pod2usage(-msg => "--output-genome-bam cannot be specified if --no-bam-output is specified!\n", -exitval => 2, -verbose => 2) if ($genGenomeBamF && !$genBamF);
+pod2usage(-msg => "The seed for random number generator must be a non-negative 32bit integer!\n", -exitval => 2, -verbose => 2) if (($seed ne "NULL") && ($seed < 0 || $seed > 0xffffffff));
if ($L < 25) { print "Warning: the seed length set is less than 25! This is only allowed if the references are not added poly(A) tails.\n"; }
$sampleName = $ARGV[3];
}
+if (((-e "$refName.ta") && !(-e "$refName.gt")) || (!(-e "$refName.ta") && (-e "$refName.gt"))) {
+ print "Allele-specific expression related reference files are corrupted!\n";
+ exit(-1);
+}
+
+$alleleS = (-e "$refName.ta") && (-e "$refName.gt");
+
if ($genGenomeBamF) {
open(INPUT, "$refName.ti");
my $line = <INPUT>; chomp($line);
}
# pipe to samtools to generate a BAM file
- $command .= " | $dir\sam/samtools view -S -b -o $imdName.bam -";
+ $command .= " | samtools view -S -b -o $imdName.bam -";
}
else {
$command = $bowtie2_path."bowtie2";
}
# pipe to samtools to generate a BAM file
- $command .= " | $dir\sam/samtools view -S -b -o $imdName.bam -";
+ $command .= " | samtools view -S -b -o $imdName.bam -";
}
if ($mTime) { $time_start = time(); }
if ($mTime) { $time_start = time(); }
-$command = $dir."rsem-parse-alignments $refName $imdName $statName";
+$command = "rsem-parse-alignments $refName $imdName $statName";
my $samInpType;
if ($is_sam) { $samInpType = "s"; }
&runCommand($command);
-$command = $dir."rsem-build-read-index $gap";
+$command = "rsem-build-read-index $gap";
if ($read_type == 0) { $command .= " 0 $quiet $imdName\_alignable.fa"; }
elsif ($read_type == 1) { $command .= " 1 $quiet $imdName\_alignable.fq"; }
elsif ($read_type == 2) { $command .= " 0 $quiet $imdName\_alignable_1.fa $imdName\_alignable_2.fa"; }
print OUTPUT "$L\n";
close(OUTPUT);
-$command = $dir."rsem-run-em $refName $read_type $sampleName $imdName $statName -p $nThreads";
+my @seeds = ();
+if ($seed ne "NULL") {
+ srand($seed);
+ for (my $i = 0; $i < 3; $i++) {
+ push(@seeds, int(rand(1 << 32)));
+ }
+}
+
+$command = "rsem-run-em $refName $read_type $sampleName $imdName $statName -p $nThreads";
if ($genBamF) {
$command .= " -b $samInpType $inpF";
if ($fn_list ne "") { $command .= " 1 $fn_list"; }
else { $command .= " 0"; }
if ($sampling) { $command .= " --sampling"; }
+ if ($seed ne "NULL") { $command .= " --seed $seeds[0]"; }
}
-if ($calcCI || $var_opt) { $command .= " --gibbs-out"; }
+if ($calcPME || $calcCI) { $command .= " --gibbs-out"; }
if ($quiet) { $command .= " -q"; }
&runCommand($command);
-&collectResults("isoform", "$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
-&collectResults("gene", "$imdName.gene_res", "$sampleName.genes.results"); # gene level
+if ($alleleS) {
+ &collectResults("allele", "$imdName.allele_res", "$sampleName.alleles.results"); # allele level
+ &collectResults("isoform", "$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
+ &collectResults("gene", "$imdName.gene_res", "$sampleName.genes.results"); # gene level
+}
+else {
+ &collectResults("isoform", "$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
+ &collectResults("gene", "$imdName.gene_res", "$sampleName.genes.results"); # gene level
+}
if ($genBamF) {
- $command = $dir."sam/samtools sort $sampleName.transcript.bam $sampleName.transcript.sorted";
+ $command = "samtools sort -@ $nThreads -m $SortMem $sampleName.transcript.bam $sampleName.transcript.sorted";
&runCommand($command);
- $command = $dir."sam/samtools index $sampleName.transcript.sorted.bam";
+ $command = "samtools index $sampleName.transcript.sorted.bam";
&runCommand($command);
if ($genGenomeBamF) {
- $command = $dir."rsem-tbam2gbam $refName $sampleName.transcript.bam $sampleName.genome.bam";
+ $command = "rsem-tbam2gbam $refName $sampleName.transcript.bam $sampleName.genome.bam";
&runCommand($command);
- $command = $dir."sam/samtools sort $sampleName.genome.bam $sampleName.genome.sorted";
+ $command = "samtools sort -@ $nThreads -m $SortMem $sampleName.genome.bam $sampleName.genome.sorted";
&runCommand($command);
- $command = $dir."sam/samtools index $sampleName.genome.sorted.bam";
+ $command = "samtools index $sampleName.genome.sorted.bam";
&runCommand($command);
}
}
if ($mTime) { $time_start = time(); }
-if ($calcCI || $var_opt) {
- $command = $dir."rsem-run-gibbs $refName $imdName $statName $BURNIN $NCV $SAMPLEGAP";
+if ($calcPME || $calcCI ) {
+ $command = "rsem-run-gibbs $refName $imdName $statName $BURNIN $NCV $SAMPLEGAP";
$command .= " -p $nThreads";
- if ($var_opt) { $command .= " --var"; }
+ if ($seed ne "NULL") { $command .= " --seed $seeds[1]"; }
if ($quiet) { $command .= " -q"; }
&runCommand($command);
}
-if ($calcCI) {
- system("mv $sampleName.isoforms.results $imdName.isoforms.results.bak1");
- system("mv $sampleName.genes.results $imdName.genes.results.bak1");
- &collectResults("isoform", "$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
- &collectResults("gene", "$imdName.gene_res", "$sampleName.genes.results"); # gene level
+if ($calcPME || $calcCI) {
+ if ($alleleS) {
+ system("mv $sampleName.alleles.results $imdName.alleles.results.bak1");
+ system("mv $sampleName.isoforms.results $imdName.isoforms.results.bak1");
+ system("mv $sampleName.genes.results $imdName.genes.results.bak1");
+ &collectResults("allele", "$imdName.allele_res", "$sampleName.alleles.results"); # allele level
+ &collectResults("isoform", "$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
+ &collectResults("gene", "$imdName.gene_res", "$sampleName.genes.results"); # gene level
+ }
+ else {
+ system("mv $sampleName.isoforms.results $imdName.isoforms.results.bak1");
+ system("mv $sampleName.genes.results $imdName.genes.results.bak1");
+ &collectResults("isoform", "$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
+ &collectResults("gene", "$imdName.gene_res", "$sampleName.genes.results"); # gene level
+ }
+}
- $command = $dir."rsem-calculate-credibility-intervals $refName $imdName $statName $CONFIDENCE $NCV $NSPC $NMB";
+if ($calcCI) {
+ $command = "rsem-calculate-credibility-intervals $refName $imdName $statName $CONFIDENCE $NCV $NSPC $NMB";
$command .= " -p $nThreads";
+ if ($seed ne "NULL") { $command .= " --seed $seeds[2]"; }
if ($quiet) { $command .= " -q"; }
&runCommand($command);
- system("mv $sampleName.isoforms.results $imdName.isoforms.results.bak2");
- system("mv $sampleName.genes.results $imdName.genes.results.bak2");
- &collectResults("isoform", "$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
- &collectResults("gene", "$imdName.gene_res", "$sampleName.genes.results"); # gene level
+ if ($alleleS) {
+ system("mv $sampleName.alleles.results $imdName.alleles.results.bak2");
+ system("mv $sampleName.isoforms.results $imdName.isoforms.results.bak2");
+ system("mv $sampleName.genes.results $imdName.genes.results.bak2");
+ &collectResults("allele", "$imdName.allele_res", "$sampleName.alleles.results"); # allele level
+ &collectResults("isoform", "$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
+ &collectResults("gene", "$imdName.gene_res", "$sampleName.genes.results"); # gene level
+ }
+ else {
+ system("mv $sampleName.isoforms.results $imdName.isoforms.results.bak2");
+ system("mv $sampleName.genes.results $imdName.genes.results.bak2");
+ &collectResults("isoform", "$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
+ &collectResults("gene", "$imdName.gene_res", "$sampleName.genes.results"); # gene level
+ }
}
if ($mTime) { $time_end = time(); $time_ci = $time_end - $time_start; }
print OUTPUT "Aligning reads: $time_alignment s.\n";
print OUTPUT "Estimating expression levels: $time_rsem s.\n";
print OUTPUT "Calculating credibility intervals: $time_ci s.\n";
- my $time_del = $time_end - $time_start;
+# my $time_del = $time_end - $time_start;
# print OUTPUT "Delete: $time_del s.\n";
close(OUTPUT);
}
=item B<-p/--num-threads> <int>
-Number of threads to use. Both Bowtie/Bowtie2 and expression estimation will use this many threads. (Default: 1)
+Number of threads to use. Both Bowtie/Bowtie2, expression estimation and 'samtools sort' will use this many threads. (Default: 1)
=item B<--no-bam-output>
When RSEM generates a BAM file, instead of outputing all alignments a read has with their posterior probabilities, one alignment is sampled according to the posterior probabilities. The sampling procedure includes the alignment to the "noise" transcript, which does not appear in the BAM file. Only the sampled alignment has a weight of 1. All other alignments have weight 0. If the "noise" transcript is sampled, all alignments appeared in the BAM file should have weight 0. (Default: off)
+=item B<--seed> <uint32>
+
+Set the seed for the random number generators used in calculating posterior mean estimates and credibility intervals. The seed must be a non-negative 32 bit interger. (Default: off)
+
+=item B<--calc-pme>
+
+Run RSEM's collapsed Gibbs sampler to calculate posterior mean estimates. (Default: off)
+
=item B<--calc-ci>
-Calculate 95% credibility intervals and posterior mean estimates. (Default: off)
+Calculate 95% credibility intervals and posterior mean estimates. (Default: off)
=item B<--seed-length> <int>
=item B<--bowtie2>
-Use Bowtie 2 instead of Bowtie to align reads. Since currently RSEM does not handle indel, local and discordant alignments, the Bowtie2 parameters are set in a way to avoid those alignments. In particular, we use options '--very-sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-rate'. "-rate", the last parameter of '--score-min' is the negative value of the mismatch rate provided by option '--bowtie2-mismatch-rate'. If reads are paired-end, we additionally use options '--no-mixed' and '--no-discordant'. (Default: off)
+Use Bowtie 2 instead of Bowtie to align reads. Since currently RSEM does not handle indel, local and discordant alignments, the Bowtie2 parameters are set in a way to avoid those alignments. In particular, we use options '--sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.1' by default. "-0.1", the last parameter of '--score-min' is the negative value of the maximum mismatch rate allowed. This rate can be set by option '--bowtie2-mismatch-rate'. If reads are paired-end, we additionally use options '--no-mixed' and '--no-discordant'. (Default: off)
=item B<--bowtie2-path> <path>
Maximum size (in memory, MB) of the auxiliary buffer used for computing credibility intervals (CI). Set it larger for a faster CI calculation. However, leaving 2 GB memory free for other usage is recommended. (Default: 1024)
+=item B<--samtools-sort-mem> <string>
+
+Set the maximum memory per thread that can be used by 'samtools sort'. <string> represents the memory and accepts suffices 'K/M/G'. RSEM will pass <string> to the '-m' option of 'samtools sort'. Please note that the default used here is different from the default used by samtools. (Default: 1G)
+
=item B<--keep-intermediate-files>
Keep temporary files generated by RSEM. RSEM creates a temporary directory, 'sample_name.temp', into which it puts all intermediate output files. If this directory already exists, RSEM overwrites all files generated by previous RSEM runs inside of it. By default, after RSEM finishes, the temporary directory is deleted. Set this option to prevent the deletion of this directory and the intermediate files inside of it. (Default: off)
contains column names separated by the tab character. The format of
each line in the rest of this file is:
-transcript_id gene_id length effective_length expected_count TPM FPKM IsoPct [pme_expected_count pme_TPM pme_FPKM IsoPct_from_pme_TPM TPM_ci_lower_bound TPM_ci_upper_bound FPKM_ci_lower_bound FPKM_ci_upper_bound]
+transcript_id gene_id length effective_length expected_count TPM FPKM IsoPct [posterior_mean_count posterior_standard_deviation_of_count pme_TPM pme_FPKM IsoPct_from_pme_TPM TPM_ci_lower_bound TPM_ci_upper_bound FPKM_ci_lower_bound FPKM_ci_upper_bound]
-Fields are separated by the tab character. Fields within "[]" are only
-presented if '--calc-ci' is set.
+Fields are separated by the tab character. Fields within "[]" are
+optional. They will not be presented if neither '--calc-pme' nor
+'--calc-ci' is set.
'transcript_id' is the transcript name of this transcript. 'gene_id'
is the gene name of the gene which this transcript belongs to (denote
gene has only one isoform or the gene information is not provided,
this field will be set to 100.
-'pme_expected_count', 'pme_TPM', 'pme_FPKM' are posterior mean
-estimates calculated by RSEM's Gibbs sampler. 'IsoPct_from_pme_TPM' is
-the isoform percentage calculated from 'pme_TPM' values.
+'posterior_mean_count', 'pme_TPM', 'pme_FPKM' are posterior mean
+estimates calculated by RSEM's Gibbs
+sampler. 'posterior_standard_deviation_of_count' is the posterior
+standard deviation of counts. 'IsoPct_from_pme_TPM' is the isoform
+percentage calculated from 'pme_TPM' values.
'TPM_ci_lower_bound', 'TPM_ci_upper_bound', 'FPKM_ci_lower_bound' and
'FPKM_ci_upper_bound' are lower(l) and upper(u) bounds of 95%
contains column names separated by the tab character. The format of
each line in the rest of this file is:
-gene_id transcript_id(s) length effective_length expected_count TPM FPKM [pme_expected_count pme_TPM pme_FPKM TPM_ci_lower_bound TPM_ci_upper_bound FPKM_ci_lower_bound FPKM_ci_upper_bound]
+gene_id transcript_id(s) length effective_length expected_count TPM FPKM [posterior_mean_count posterior_standard_deviation_of_count pme_TPM pme_FPKM TPM_ci_lower_bound TPM_ci_upper_bound FPKM_ci_lower_bound FPKM_ci_upper_bound]
-Fields are separated by the tab character. Fields within "[]" are only
-presented if '--calc-ci' is set.
+Fields are separated by the tab character. Fields within "[]" are
+optional. They will not be presented if neither '--calc-pme' nor
+'--calc-ci' is set.
'transcript_id(s)' is a comma-separated list of transcript_ids
belonging to this gene. If no gene information is provided, 'gene_id'
effective lengths (weighted by 'IsoPct'). A gene's abundance estimates
are just the sum of its transcripts' abundance estimates.
+=item B<sample_name.alleles.results>
+
+Only generated when the RSEM references are built with allele-specific
+transcripts.
+
+This file contains allele level expression estimates for
+allele-specific expression calculation. The first line
+contains column names separated by the tab character. The format of
+each line in the rest of this file is:
+
+allele_id transcript_id gene_id length effective_length expected_count TPM FPKM AlleleIsoPct AlleleGenePct [posterior_mean_count posterior_standard_deviation_of_count pme_TPM pme_FPKM AlleleIsoPct_from_pme_TPM AlleleGenePct_from_pme_TPM TPM_ci_lower_bound TPM_ci_upper_bound FPKM_ci_lower_bound FPKM_ci_upper_bound]
+
+Fields are separated by the tab character. Fields within "[]" are
+optional. They will not be presented if neither '--calc-pme' nor
+'--calc-ci' is set.
+
+'allele_id' is the allele-specific name of this allele-specific transcript.
+
+'AlleleIsoPct' stands for allele-specific percentage on isoform
+level. It is the percentage of this allele-specific transcript's
+abundance over its parent transcript's abundance. If its parent
+transcript has only one allele variant form, this field will be set to
+100.
+
+'AlleleGenePct' stands for allele-specific percentage on gene
+level. It is the percentage of this allele-specific transcript's
+abundance over its parent gene's abundance.
+
+'AlleleIsoPct_from_pme_TPM' and 'AlleleGenePct_from_pme_TPM' have
+similar meanings. They are calculated based on posterior mean
+estimates.
+
+Please note that if this file is present, the fields 'length' and
+'effective_length' in 'sample_name.isoforms.results' should be
+interpreted similarly as the corresponding definitions in
+'sample_name.genes.results'.
+
=item B<sample_name.transcript.bam, sample_name.transcript.sorted.bam and sample_name.transcript.sorted.bam.bai>
Only generated when --no-bam-output is not specified.
projects / rsem.git / blobdiff