my $NMB = 1024; # default
-my $status;
+my $status = 0;
my $read_type = 1; # default, single end with qual
my $B = 20;
my $nThreads = 1;
-my $genBamF = 0;
+my $genBamF = 1; # default is generating transcript bam file
+my $genGenomeBamF = 0;
+my $sampling = 0;
my $calcCI = 0;
my $quiet = 0;
my $help = 0;
my $strand_specific = 0;
+my $mTime = 0;
+my ($time_start, $time_end, $time_alignment, $time_rsem, $time_ci) = (0, 0, 0, 0, 0);
+
GetOptions("keep-intermediate-files" => \$keep_intermediate_files,
"no-qualities" => \$no_qual,
"paired-end" => \$paired_end,
"estimate-rspd" => \$estRSPD,
"num-rspd-bins=i" => \$B,
"p|num-threads=i" => \$nThreads,
- "out-bam" => \$genBamF,
+ "output-genome-bam" => \$genGenomeBamF,
+ "sampling-for-bam" => \$sampling,
"calc-ci" => \$calcCI,
"ci-memory=i" => \$NMB,
+ "time" => \$mTime,
"q|quiet" => \$quiet,
"h|help" => \$help) or pod2usage(-exitval => 2, -verbose => 2);
pod2usage(-msg => "Min fragment length should be smaller or equal to max fragment length!", -exitval => 2, -verbose => 2) if ($minL > $maxL);
pod2usage(-msg => "The memory allocated for calculating credibility intervals should be at least 1 MB!\n", -exitval => 2, -verbose => 2) if ($NMB < 1);
pod2usage(-msg => "Number of threads should be at least 1!\n", -exitval => 2, -verbose => 2) if ($nThreads < 1);
+pod2usage(-msg => "Seed length should be at least 25!\n", -exitval => 2, -verbose => 2) if ($L < 25);
+pod2usage(-msg => "--sampling-for-bam cannot be specified if --out-bam is not specified!\n", -exitval => 2, -verbose => 2) if ($sampling && !$genBamF);
if ($strand_specific) { $probF = 1.0; }
$sampleName = $ARGV[3];
}
+if ($genGenomeBamF) {
+ open(INPUT, "$refName.ti");
+ my $line = <INPUT>; chomp($line);
+ close(INPUT);
+ my ($M, $type) = split(/ /, $line);
+ pod2usage(-msg => "No genome information provided, so genome bam file cannot be generated!\n", -exitval => 2, -verbose => 2) if ($type != 0);
+}
+
my $pos = rindex($sampleName, '/');
if ($pos < 0) { $sampleToken = $sampleName; }
else { $sampleToken = substr($sampleName, $pos + 1); }
}
$command .= " | gzip > $imdName.sam.gz";
- print "$command\n";
- $status = system($command);
- if ($status != 0) {
- print "bowtie failed! Please check if you provide correct parameters/options for the pipeline!\n";
- exit(-1);
- }
- print "\n";
+
+ if ($mTime) { $time_start = time(); }
+
+ &runCommand($command);
+
+ if ($mTime) { $time_end = time(); $time_alignment = $time_end - $time_start; }
$inpF = "$imdName.sam.gz";
$is_sam = 1; # output of bowtie is a sam file
}
+if ($mTime) { $time_start = time(); }
+
$command = $dir."rsem-parse-alignments $refName $sampleName $sampleToken";
my $samInpType;
if ($tagName ne "") { $command .= " -tag $tagName"; }
if ($quiet) { $command .= " -q"; }
-print "$command\n";
-$status = system($command);
-if ($status != 0) {
- print "rsem-parse-alignments failed! Please check if you provide correct parameters/options for the pipeline!\n";
- exit(-1);
-}
-print "\n";
+&runCommand($command);
$command = $dir."rsem-build-read-index $gap";
switch($read_type) {
case 2 { $command .= " 0 $quiet $imdName\_alignable_1.fa $imdName\_alignable_2.fa"; }
case 3 { $command .= " 1 $quiet $imdName\_alignable_1.fq $imdName\_alignable_2.fq"; }
}
-print "$command\n";
-$status = system($command);
-if ($status != 0) {
- print "rsem-build-read-index failed! Please check if you provide correct parameters/options for the pipeline!\n";
- exit(-1);
-}
-print "\n";
+&runCommand($command);
-$status = open(OUTPUT, ">$imdName.mparams");
-if ($status == 0) { print "Cannot generate $imdName.mparams!\n"; exit(-1); }
+my $doesOpen = open(OUTPUT, ">$imdName.mparams");
+if ($doesOpen == 0) { print "Cannot generate $imdName.mparams!\n"; exit(-1); }
print OUTPUT "$minL $maxL\n";
print OUTPUT "$probF\n";
print OUTPUT "$estRSPD\n";
$command .= " -b $samInpType $inpF";
if ($fn_list ne "") { $command .= " 1 $fn_list"; }
else { $command .= " 0"; }
+ if ($sampling) { $command .= " --sampling"; }
}
if ($calcCI) { $command .= " --gibbs-out"; }
if ($quiet) { $command .= " -q"; }
-print "$command\n";
-$status = system($command);
-if ($status != 0) {
- print "rsem-run-em failed! Please check if you provide correct parameters/options for the pipeline!\n";
- exit(-1);
-}
-print "\n";
+&runCommand($command);
if ($genBamF) {
- $command = $dir."sam/samtools sort $sampleName.bam $sampleName.sorted";
- print "$command\n";
- $status = system($command);
- if ($status != 0) {
- print "sam/samtools sort failed! Please check if you provide correct parameters/options for the pipeline!\n";
- exit(-1);
- }
- print "\n";
- $command = $dir."sam/samtools index $sampleName.sorted.bam";
- print "$command\n";
- $status = system($command);
- if ($status != 0) {
- print "sam/samtools index failed! Please check if you provide correct parameters/options for the pipeline!\n";
- exit(-1);
+ $command = $dir."sam/samtools sort $sampleName.transcript.bam $sampleName.transcript.sorted";
+ &runCommand($command);
+ $command = $dir."sam/samtools index $sampleName.transcript.sorted.bam";
+ &runCommand($command);
+
+ if ($genGenomeBamF) {
+ $command = $dir."rsem-tbam2gbam $refName $sampleName.transcript.bam $sampleName.genome.bam";
+ &runCommand($command);
+ $command = $dir."sam/samtools sort $sampleName.genome.bam $sampleName.genome.sorted";
+ &runCommand($command);
+ $command = $dir."sam/samtools index $sampleName.genome.sorted.bam";
+ &runCommand($command);
}
- print "\n";
}
&collectResults("$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
&collectResults("$imdName.gene_res", "$sampleName.genes.results"); # gene level
+if ($mTime) { $time_end = time(); $time_rsem = $time_end - $time_start; }
+
+if ($mTime) { $time_start = time(); }
+
if ($calcCI) {
$command = $dir."rsem-run-gibbs $refName $sampleName $sampleToken $BURNIN $CHAINLEN $SAMPLEGAP";
# $command .= " -p $nThreads";
if ($quiet) { $command .= " -q"; }
- print "$command\n";
- $status = system($command);
- if ($status != 0) {
- print "rsem-run-gibbs failed! Please check if you provide correct parameters/options for the pipeline!\n";
- exit(-1);
- }
- print "\n";
+ &runCommand($command);
system("mv $sampleName.isoforms.results $imdName.isoforms.results.bak1");
system("mv $sampleName.genes.results $imdName.genes.results.bak1");
$command = $dir."rsem-calculate-credibility-intervals $refName $sampleName $sampleToken $CONFIDENCE $NSPC $NMB";
if ($quiet) { $command .= " -q"; }
- print "$command\n";
- $status = system($command);
- if ($status != 0) {
- print "rsem-calculate-credibility-intervals failed! Please check if you provide correct parameters/options for the pipeline!\n";
- exit(-1);
- }
- print "\n";
+ &runCommand($command);
system("mv $sampleName.isoforms.results $imdName.isoforms.results.bak2");
system("mv $sampleName.genes.results $imdName.genes.results.bak2");
&collectResults("$imdName.gene_res", "$sampleName.genes.results"); # gene level
}
+if ($mTime) { $time_end = time(); $time_ci = $time_end - $time_start; }
+
+if ($mTime) { $time_start = time(); }
+
if (!$keep_intermediate_files) {
- $status = system ("rm -rf $temp_dir");
- if ($status != 0) {
- print "Fail to delete the temporary folder!\n";
+ &runCommand("rm -rf $temp_dir", "Fail to delete the temporary folder!");
+}
+
+if ($mTime) { $time_end = time(); }
+
+if ($mTime) {
+ open(OUTPUT, ">$sampleName.time");
+ print OUTPUT "Alignment: $time_alignment s.\n";
+ print OUTPUT "RSEM: $time_rsem s.\n";
+ print OUTPUT "CI: $time_ci s.\n";
+ my $time_del = $time_end - $time_start;
+ print OUTPUT "Delete: $time_del s.\n";
+ close(OUTPUT);
+}
+
+# command, {err_msg}
+sub runCommand {
+ print $_[0]."\n";
+ my $status = system($_[0]);
+ if ($status != 0) {
+ my $errmsg;
+ if (scalar(@_) > 1) { $errmsg = $_[1]; }
+ else { $errmsg = "$command failed! Plase check if you provide correct parameters/options for the pipeline!"; }
+ print $errmsg."\n";
exit(-1);
}
+ print "\n";
}
# inpF, outF
Number of threads to use. Both Bowtie and expression estimation will use this many threads. (Default: 1)
-=item B<--out-bam>
+=item B<--output-genome-bam>
+
+Generate a BAM file, 'sample_name.genome.bam', with alignments mapped to genomic coordinates and annotated with their posterior probabilities. In addition, RSEM will call samtools (included in RSEM package) to sort and index the bam file. 'sample_name.genome.sorted.bam' and 'sample_name.genome.sorted.bam.bai' will be generated. (Default: off)
-Generate a BAM file, 'sample_name.bam', with alignments mapped to genomic coordinates and annotated with their posterior probabilities. In addition, RSEM will call samtools (included in RSEM package) to sort and index the bam file. 'sample_name.sorted.bam' and 'sample_name.sorted.bam.bai' will be generated. (Default: off)
+=item B<--sampling-for-bam>
+When RSEM generates a BAM file, instead of outputing all alignments a read has with their posterior probabilities, one alignment is sampled and outputed according to the posterior probabilities. If the sampling result is that the read comes from the "noise" transcript, nothing is outputed. (Default: off)
+
=item B<--calc-ci>
Calculate 95% credibility intervals and posterior mean estimates. (Default: off)
=item B<--seed-length> <int>
-Seed length used by the read aligner. Providing the correct value for this parameter is important for RSEM's accuracy if the data are single-end reads. If RSEM runs Bowtie, it uses this value for Bowtie's seed length parameter. (Default: 25)
+Seed length used by the read aligner. Providing the correct value for this parameter is important for RSEM's accuracy if the data are single-end reads. If RSEM runs Bowtie, it uses this value for Bowtie's seed length parameter. The minimum value is 25. Any read with its or at least one of its mates' (for paired-end reads) length less than 25 will be ignored. (Default: 25)
=item B<--tag> <string>
Input quality scores are encoded as Phred+33. (Default: on)
=item B<--phred64-quals>
-
+
Input quality scores are encoded as Phred+64 (default for GA Pipeline ver. >= 1.3). (Default: off)
=item B<--solexa-quals>
-
+
Input quality scores are solexa encoded (from GA Pipeline ver. < 1.3). (Default: off)
=item B<--forward-prob> <double>
=head1 DESCRIPTION
-In its default mode, this program aligns input reads against a reference transcriptome with Bowtie and calculates expression values using the alignments. RSEM assumes the data are single-end reads with quality scores, unless the '--paired-end' or '--no-qualities' options are specified. Users may use an alternative aligner by specifying one of the --sam and --bam options, and providing an alignment file in the specified format. However, users should make sure the alignment file satisfies the requirements mentioned in ARGUMENTS section.
+In its default mode, this program aligns input reads against a reference transcriptome with Bowtie and calculates expression values using the alignments. RSEM assumes the data are single-end reads with quality scores, unless the '--paired-end' or '--no-qualities' options are specified. Users may use an alternative aligner by specifying one of the --sam and --bam options, and providing an alignment file in the specified format. However, users should make sure that they align against the indices generated by 'rsem-prepare-reference' and the alignment file satisfies the requirements mentioned in ARGUMENTS section.
One simple way to make the alignment file (e.g. input.sam) satisfying RSEM's requirements (assuming the aligner used put mates in a paired-end read adjacent) is to use the following command:
sort -k 1,1 -s input.sam > input.sorted.sam
-The SAM/BAM format RSEM uses is v1.3. However, it is compatible with old SAM/BAM format.
+The SAM/BAM format RSEM uses is v1.4. However, it is compatible with old SAM/BAM format.
The user must run 'rsem-prepare-reference' with the appropriate reference before using this program.
'rsem-prepare-reference', there will be no tab after the
tau_value field.
-=item B<sample_name.bam, sample_name.sorted.bam and sample_name.sorted.bam.bai>
+=item B<sample_name.transcript.bam, sample_name.transcript.sorted.bam and sample_name.transcript.sorted.bam.bai>
+
+'sample_name.transcript.bam' is a BAM-formatted file of read
+alignments in transcript coordinates. The MAPQ field of each alignment
+is set to min(100, floor(-10 * log10(1.0 - w) + 0.5)), where w is the
+posterior probability of that alignment being the true mapping of a
+read. In addition, RSEM pads a new tag ZW:f:value, where value is a
+single precision floating number representing the posterior
+probability.
+
+'sample_name.transcript.sorted.bam' and
+'sample_name.transcript.sorted.bam.bai' are the sorted BAM file and
+indices generated by samtools (included in RSEM package).
+
+=item B<sample_name.genome.bam, sample_name.genome.sorted.bam and sample_name.genome.sorted.bam.bai>
-Only generated when --out-bam is specified.
+Only generated when --output-genome-bam is specified.
-'sample_name.bam' is a BAM-formatted file of read alignments in
+'sample_name.genome.bam' is a BAM-formatted file of read alignments in
genomic coordinates. Alignments of reads that have identical genomic
coordinates (i.e., alignments to different isoforms that share the
same genomic region) are collapsed into one alignment. The MAPQ field
-of each alignment is set to max(100, floor(-10 * log10(1.0 - w) +
+of each alignment is set to min(100, floor(-10 * log10(1.0 - w) +
0.5)), where w is the posterior probability of that alignment being
the true mapping of a read. In addition, RSEM pads a new tag
ZW:f:value, where value is a single precision floating number
representing the posterior probability.
-'sample_name.sorted.bam' and 'sample_name.sorted.bam.bai' are the
+'sample_name.genome.sorted.bam' and 'sample_name.genome.sorted.bam.bai' are the
sorted BAM file and indices generated by samtools (included in RSEM package).
=item B<sample_name.stat>
Assume the path to the bowtie executables is in the user's PATH environment variable. Reference files are under '/ref' with name 'mm9'.
-1) '/data/mmliver.fq', single-end reads with quality scores. Quality scores are encoded as for 'GA pipeline version >= 1.3'. We want to use 8 threads and generate a BAM file:
+1) '/data/mmliver.fq', single-end reads with quality scores. Quality scores are encoded as for 'GA pipeline version >= 1.3'. We want to use 8 threads and generate a genome BAM file:
rsem-calculate-expression --phred64-quals \
-p 8 \
- --out-bam \
+ --output-genome-bam \
/data/mmliver.fq \
/ref/mm9 \
mmliver_single_quals
-2) '/data/mmliver_1.fq' and '/data/mmliver_2.fq', paired-end reads with quality scores. Quality scores are in SANGER format. We want to use 8 threads and do not generate a BAM file:
+2) '/data/mmliver_1.fq' and '/data/mmliver_2.fq', paired-end reads with quality scores. Quality scores are in SANGER format. We want to use 8 threads and do not generate a genome BAM file:
rsem-calculate-expression -p 8 \
--paired-end \
/ref/mm9 \
mmliver_paired_end_quals
-3) '/data/mmliver.fa', single-end reads without quality scores. We want to use 8 threads and generate a BAM file:
+3) '/data/mmliver.fa', single-end reads without quality scores. We want to use 8 threads:
rsem-calculate-expression -p 8 \
--no-qualities \
/ref/mm9 \
mmliver_single_without_quals
-4) Data are the same as 1). We want to take a fragment length distribution into consideration. We set the fragment length mean to 150 and the standard deviation to 35. In addition to a BAM file, we also want to generate credibility intervals. We allow RSEM to use 1GB of memory for CI calculation.
+4) Data are the same as 1). We want to take a fragment length distribution into consideration. We set the fragment length mean to 150 and the standard deviation to 35. In addition to a BAM file, we also want to generate credibility intervals. We allow RSEM to use 1GB of memory for CI calculation:
rsem-calculate-expression --bowtie-path /sw/bowtie \
--phred64-quals \
--fragment-length-mean 150.0 \
--fragment-length-sd 35.0 \
-p 8 \
- --out-bam \
+ --output-genome-bam \
--calc-ci \
--ci-memory 1024 \
/data/mmliver.fq \
/ref/mm9 \
mmliver_single_quals
-5) '/data/mmliver_paired_end_quals.bam', paired-end reads with quality scores. We want to use 8 threads and do not generate a BAM file:
+5) '/data/mmliver_paired_end_quals.bam', paired-end reads with quality scores. We want to use 8 threads:
rsem-calculate-expression --paired-end \
--bam \
=cut
-# LocalWords: usr
projects / rsem.git / blobdiff