13 my $CONFIDENCE = 0.95;
16 my $NMB = 1024; # default
20 my $read_type = 1; # default, single end with qual
54 my $keep_intermediate_files = 0;
56 my $strand_specific = 0;
59 my ($time_start, $time_end, $time_alignment, $time_rsem, $time_ci) = (0, 0, 0, 0, 0);
61 GetOptions("keep-intermediate-files" => \$keep_intermediate_files,
62 "no-qualities" => \$no_qual,
63 "paired-end" => \$paired_end,
64 "strand-specific" => \$strand_specific,
67 "sam-header-info=s" => \$fn_list,
69 "seed-length=i" => \$L,
70 "bowtie-path=s" => \$bowtie_path,
73 "bowtie-m=i" => \$maxHits,
74 "phred33-quals" => \$phred33,
75 "phred64-quals" => \$phred64, #solexa1.3-quals" => \$phred64,
76 "solexa-quals" => \$solexa,
77 "forward-prob=f" => \$probF,
78 "fragment-length-min=i" => \$minL,
79 "fragment-length-max=i" => \$maxL,
80 "fragment-length-mean=f" => \$mean,
81 "fragment-length-sd=f" => \$sd,
82 "estimate-rspd" => \$estRSPD,
83 "num-rspd-bins=i" => \$B,
84 "p|num-threads=i" => \$nThreads,
85 "out-bam" => \$genBamF,
86 "calc-ci" => \$calcCI,
87 "ci-memory=i" => \$NMB,
90 "h|help" => \$help) or pod2usage(-exitval => 2, -verbose => 2);
92 pod2usage(-verbose => 2) if ($help == 1);
95 #check parameters and options
97 if ($is_sam || $is_bam) {
98 pod2usage(-msg => "Invalid number of arguments!", -exitval => 2, -verbose => 2) if (scalar(@ARGV) != 3);
99 pod2usage(-msg => "--sam and --bam cannot be active at the same time!", -exitval => 2, -verbose => 2) if ($is_sam == 1&& $is_bam == 1);
100 pod2usage(-msg => "--bowtie-path, --bowtie-n, --bowtie-e, --bowtie-m, --phred33-quals, --phred64-quals or --solexa-quals cannot be set if input is SAM/BAM format!", -exitval => 2, -verbose => 2) if ($bowtie_path ne "" || $C != 2 || $E != 99999999 || $maxHits != 200 || $phred33 || $phred64 || $solexa);
103 pod2usage(-msg => "Invalid number of arguments!", -exitval => 2, -verbose => 2) if (!$paired_end && scalar(@ARGV) != 3 || $paired_end && scalar(@ARGV) != 4);
104 pod2usage(-msg => "Only one of --phred33-quals --phred64-quals/--solexa1.3-quals --solexa-suqls can be active!", -exitval => 2, -verbose => 2) if ($phred33 + $phred64 + $solexa > 1);
105 podwusage(-msg => "--sam , --bam or --sam-header-info cannot be set if use bowtie aligner to produce alignments!", -exitval => 2, -verbose => 2) if ($is_sam || $is_bam || $fn_list ne "");
108 pod2usage(-msg => "Forward probability should be in [0, 1]!", -exitval => 2, -verbose => 2) if ($probF < 0 || $probF > 1);
109 pod2usage(-msg => "Min fragment length should be at least 1!", -exitval => 2, -verbose => 2) if ($minL < 1);
110 pod2usage(-msg => "Min fragment length should be smaller or equal to max fragment length!", -exitval => 2, -verbose => 2) if ($minL > $maxL);
111 pod2usage(-msg => "The memory allocated for calculating credibility intervals should be at least 1 MB!\n", -exitval => 2, -verbose => 2) if ($NMB < 1);
112 pod2usage(-msg => "Number of threads should be at least 1!\n", -exitval => 2, -verbose => 2) if ($nThreads < 1);
114 if ($strand_specific) { $probF = 1.0; }
120 my ($refName, $sampleName, $sampleToken, $temp_dir, $stat_dir, $imdName) = ();
124 if ($no_qual) { $read_type = 2; }
125 else { $read_type = 3; }
128 if ($no_qual) { $read_type = 0; }
129 else { $read_type = 1; }
132 if (scalar(@ARGV) == 3) {
133 if ($is_sam || $is_bam) { $inpF = $ARGV[0]; }
134 else {$mate1_list = $ARGV[0]; }
136 $sampleName = $ARGV[2];
139 $mate1_list = $ARGV[0];
140 $mate2_list = $ARGV[1];
142 $sampleName = $ARGV[3];
145 my $pos = rindex($sampleName, '/');
146 if ($pos < 0) { $sampleToken = $sampleName; }
147 else { $sampleToken = substr($sampleName, $pos + 1); }
149 $temp_dir = "$sampleName.temp";
150 $stat_dir = "$sampleName.stat";
152 if (!(-d $temp_dir) && !mkdir($temp_dir)) { print "Fail to create folder $temp_dir.\n"; exit(-1); }
153 if (!(-d $stat_dir) && !mkdir($stat_dir)) { print "Fail to create folder $stat_dir.\n"; exit(-1); }
155 $imdName = "$temp_dir/$sampleToken";
157 if (!$is_sam && !$is_bam && $phred33 + $phred64 + $solexa == 0) { $phred33 = 1; }
159 my ($mate_minL, $mate_maxL) = (1, $maxL);
161 if ($bowtie_path ne "") { $bowtie_path .= "/"; }
163 my ($fn, $dir, $suf) = fileparse($0);
166 if (!$is_sam && !$is_bam) {
167 $command = $bowtie_path."bowtie";
168 if ($read_type == 0 || $read_type == 2) { $command .= " -f"; }
169 else { $command .= " -q"; }
171 if ($phred33) { $command .= " --phred33-quals"; }
172 elsif ($phred64) { $command .= " --phred64-quals"; }
173 elsif ($solexa) { $command .= " --solexa-quals"; }
174 else { print "Oh, no!!!"; exit(2); }
176 $command .= " -n $C -e $E -l $L";
178 if ($read_type == 2 || $read_type == 3) { $command .= " -I $minL -X $maxL"; }
180 if ($strand_specific || $probF == 1.0) { $command .= " --norc"; }
181 elsif ($probF == 0.0) { $command .= " --nofw"; }
183 $command .= " -p $nThreads -a -m $maxHits -S";
184 if ($quiet) { $command .= " --quiet"; }
186 $command .= " $refName";
187 if ($read_type == 0 || $read_type == 1) {
188 $command .= " $mate1_list";
191 $command .= " -1 $mate1_list -2 $mate2_list";
194 $command .= " | gzip > $imdName.sam.gz";
197 if ($mTime) { $time_start = time(); }
199 $status = system($command);
201 if ($mTime) { $time_end = time(); $time_alignment = $time_end - $time_start; }
204 print "bowtie failed! Please check if you provide correct parameters/options for the pipeline!\n";
209 $inpF = "$imdName.sam.gz";
210 $is_sam = 1; # output of bowtie is a sam file
213 if ($mTime) { $time_start = time(); }
215 $command = $dir."rsem-parse-alignments $refName $sampleName $sampleToken";
218 if ($is_sam) { $samInpType = "s"; }
219 elsif ($is_bam) { $samInpType = "b"; }
221 $command .= " $samInpType $inpF -t $read_type";
222 if ($fn_list ne "") { $command .= " -l $fn_list"; }
223 if ($tagName ne "") { $command .= " -tag $tagName"; }
224 if ($quiet) { $command .= " -q"; }
227 $status = system($command);
229 print "rsem-parse-alignments failed! Please check if you provide correct parameters/options for the pipeline!\n";
234 $command = $dir."rsem-build-read-index $gap";
236 case 0 { $command .= " 0 $quiet $imdName\_alignable.fa"; }
237 case 1 { $command .= " 1 $quiet $imdName\_alignable.fq"; }
238 case 2 { $command .= " 0 $quiet $imdName\_alignable_1.fa $imdName\_alignable_2.fa"; }
239 case 3 { $command .= " 1 $quiet $imdName\_alignable_1.fq $imdName\_alignable_2.fq"; }
242 $status = system($command);
244 print "rsem-build-read-index failed! Please check if you provide correct parameters/options for the pipeline!\n";
249 my $doesOpen = open(OUTPUT, ">$imdName.mparams");
250 if ($doesOpen == 0) { print "Cannot generate $imdName.mparams!\n"; exit(-1); }
251 print OUTPUT "$minL $maxL\n";
252 print OUTPUT "$probF\n";
253 print OUTPUT "$estRSPD\n";
255 print OUTPUT "$mate_minL $mate_maxL\n";
256 print OUTPUT "$mean $sd\n";
260 $command = $dir."rsem-run-em $refName $read_type $sampleName $sampleToken -p $nThreads";
262 $command .= " -b $samInpType $inpF";
263 if ($fn_list ne "") { $command .= " 1 $fn_list"; }
264 else { $command .= " 0"; }
266 if ($calcCI) { $command .= " --gibbs-out"; }
267 if ($quiet) { $command .= " -q"; }
270 $status = system($command);
272 print "rsem-run-em failed! Please check if you provide correct parameters/options for the pipeline!\n";
278 $command = $dir."sam/samtools sort $sampleName.bam $sampleName.sorted";
280 $status = system($command);
282 print "sam/samtools sort failed! Please check if you provide correct parameters/options for the pipeline!\n";
286 $command = $dir."sam/samtools index $sampleName.sorted.bam";
288 $status = system($command);
290 print "sam/samtools index failed! Please check if you provide correct parameters/options for the pipeline!\n";
296 &collectResults("$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
297 &collectResults("$imdName.gene_res", "$sampleName.genes.results"); # gene level
299 if ($mTime) { $time_end = time(); $time_rsem = $time_end - $time_start; }
301 if ($mTime) { $time_start = time(); }
304 $command = $dir."rsem-run-gibbs $refName $sampleName $sampleToken $BURNIN $CHAINLEN $SAMPLEGAP";
305 # $command .= " -p $nThreads";
306 if ($quiet) { $command .= " -q"; }
308 $status = system($command);
310 print "rsem-run-gibbs failed! Please check if you provide correct parameters/options for the pipeline!\n";
315 system("mv $sampleName.isoforms.results $imdName.isoforms.results.bak1");
316 system("mv $sampleName.genes.results $imdName.genes.results.bak1");
317 &collectResults("$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
318 &collectResults("$imdName.gene_res", "$sampleName.genes.results"); # gene level
320 $command = $dir."rsem-calculate-credibility-intervals $refName $sampleName $sampleToken $CONFIDENCE $NSPC $NMB";
321 if ($quiet) { $command .= " -q"; }
323 $status = system($command);
325 print "rsem-calculate-credibility-intervals failed! Please check if you provide correct parameters/options for the pipeline!\n";
330 system("mv $sampleName.isoforms.results $imdName.isoforms.results.bak2");
331 system("mv $sampleName.genes.results $imdName.genes.results.bak2");
332 &collectResults("$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
333 &collectResults("$imdName.gene_res", "$sampleName.genes.results"); # gene level
336 if ($mTime) { $time_end = time(); $time_ci = $time_end - $time_start; }
338 if ($mTime) { $time_start = time(); }
340 if (!$keep_intermediate_files) {
341 $status = system("rm -rf $temp_dir");
343 print "Fail to delete the temporary folder!\n";
348 if ($mTime) { $time_end = time(); }
351 open(OUTPUT, ">$sampleName.time");
352 print OUTPUT "Alignment: $time_alignment s.\n";
353 print OUTPUT "RSEM: $time_rsem s.\n";
354 print OUTPUT "CI: $time_ci s.\n";
355 my $time_del = $time_end - $time_start;
356 print OUTPUT "Delete: $time_del s.\n";
364 my (@results, @comment) = ();
371 $local_status = open(INPUT, $inpF);
372 if ($local_status == 0) { print "Fail to open file $inpF!\n"; exit(-1); }
377 while ($line = <INPUT>) {
380 my @local_arr = split(/\t/, $line);
381 if ($cnt == 4) { @comment = @local_arr; }
382 else { push(@results, \@local_arr); }
385 push(@results, \@comment);
388 $local_status = open(OUTPUT, ">$outF");
389 if ($local_status == 0) { print "Fail to create file $outF!\n"; exit(-1); }
391 my $n = scalar(@results);
392 my $m = scalar(@{$results[0]});
393 for (my $i = 0; $i < $m; $i++) {
395 for (my $j = 0; $j < $n; $j++) { push(@out_arr, $results[$j][$i]); }
397 print OUTPUT "@out_arr\n";
407 rsem-calculate-expression
413 rsem-calculate-expression [options] upstream_read_file(s) reference_name sample_name
414 rsem-calculate-expression [options] --paired-end upstream_read_file(s) downstream_read_file(s) reference_name sample_name
415 rsem-calculate-expression [options] --sam/--bam [--paired-end] input reference_name sample_name
423 =item B<upstream_read_files(s)>
425 Comma-separated list of files containing single-end reads or upstream reads for paired-end data. By default, these files are assumed to be in FASTQ format. If the --no-qualities option is specified, then FASTA format is expected.
427 =item B<downstream_read_file(s)>
429 Comma-separated list of files containing downstream reads which are paired with the upstream reads. By default, these files are assumed to be in FASTQ format. If the --no-qualities option is specified, then FASTA format is expected.
433 SAM/BAM formatted input file. If "-" is specified for the filename, SAM/BAM input is instead assumed to come from standard input. RSEM requires all alignments of the same read group together. For paired-end reads, RSEM also requires the two mates of any alignment be adjacent. See Description section for how to make input file obey RSEM's requirements.
435 =item B<reference_name>
437 The name of the reference used. The user must have run 'rsem-prepare-reference' with this reference_name before running this program.
441 The name of the sample analyzed. All output files are prefixed by this name (e.g., sample_name.genes.results)
449 =item B<--paired-end>
451 Input reads are paired-end reads. (Default: off)
453 =item B<--no-qualities>
455 Input reads do not contain quality scores. (Default: off)
457 =item B<--strand-specific>
459 The RNA-Seq protocol used to generate the reads is strand specific, i.e., all (upstream) reads are derived from the forward strand. This option is equivalent to --forward-prob=1.0. With this option set, if RSEM runs the Bowtie aligner, the '--norc' Bowtie option will be used, which disables alignment to the reverse strand of transcripts. (Default: off)
463 Input file is in SAM format. (Default: off)
467 Input file is in BAM format. (Default: off)
469 =item B<--sam-header-info> <file>
471 RSEM reads header information from input by default. If this option is on, header information is read from the specified file. For the format of the file, please see SAM official website. (Default: "")
473 =item B<-p/--num-threads> <int>
475 Number of threads to use. Both Bowtie and expression estimation will use this many threads. (Default: 1)
479 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)
483 Calculate 95% credibility intervals and posterior mean estimates. (Default: off)
485 =item B<--seed-length> <int>
487 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)
489 =item B<--tag> <string>
491 The name of the optional field used in the SAM input for identifying a read with too many valid alignments. The field should have the format <tagName>:i:<value>, where a <value> bigger than 0 indicates a read with too many alignments. (Default: "")
493 =item B<--bowtie-path> <path>
495 The path to the bowtie executables. (Default: the path to the bowtie executables is assumed to be in the user's PATH environment variable)
497 =item B<--bowtie-n> <int>
499 (Bowtie parameter) max # of mismatches in the seed. (Range: 0-3, Default: 2)
501 =item B<--bowtie-e> <int>
503 (Bowtie parameter) max sum of mismatch quality scores across the alignment. (Default: 99999999)
505 =item B<--bowtie-m> <int>
507 (Bowtie parameter) suppress all alignments for a read if > <int> valid alignments exist. (Default: 200)
509 =item B<--phred33-quals>
511 Input quality scores are encoded as Phred+33. (Default: on)
513 =item B<--phred64-quals>
515 Input quality scores are encoded as Phred+64 (default for GA Pipeline ver. >= 1.3). (Default: off)
517 =item B<--solexa-quals>
519 Input quality scores are solexa encoded (from GA Pipeline ver. < 1.3). (Default: off)
521 =item B<--forward-prob> <double>
523 Probability of generating a read from the forward strand of a transcript. Set to 1 for a strand-specific protocol where all (upstream) reads are derived from the forward strand, 0 for a strand-specific protocol where all (upstream) read are derived from the reverse strand, or 0.5 for a non-strand-specific protocol. (Default: 0.5)
525 =item B<--fragment-length-min> <int>
527 Minimum read/insert length allowed. This is also the value for the bowtie -I option. (Default: 1)
529 =item B<--fragment-length-max> <int>
531 Maximum read/insert length allowed. This is also the value for the bowtie -X option. (Default: 1000)
533 =item B<--fragment-length-mean> <double>
535 (single-end data only) The mean of the fragment length distribution, which is assumed to be a Gaussian. (Default: -1, which disables use of the fragment length distribution)
537 =item B<--fragment-length-sd> <double>
539 (single-end data only) The standard deviation of the fragment length distribution, which is assumed to be a Gaussian. (Default: 0, which assumes that all fragments are of the same length, given by the rounded value of B<--fragment-length-mean>)
541 =item B<--estimate-rspd>
543 Set this option if you want to estimate the read start position distribution (RSPD) from data. Otherwise, RSEM will use a uniform RSPD. (Default: off)
545 =item B<--num-rspd-bins> <int>
547 Number of bins in the RSPD. Only relevant when '--estimate-rspd' is specified. Use of the default setting is recommended. (Default: 20)
549 =item B<--ci-memory> <int>
551 Amount of memory (in MB) RSEM is allowed to use for computing credibility intervals. (Default: 1024)
553 =item B<--keep-intermediate-files>
555 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)
559 Suppress the output of logging information. (Default: off)
563 Show help information.
569 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.
571 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:
573 sort -k 1,1 -s input.sam > input.sorted.sam
575 The SAM/BAM format RSEM uses is v1.3. However, it is compatible with old SAM/BAM format.
577 The user must run 'rsem-prepare-reference' with the appropriate reference before using this program.
579 For single-end data, it is strongly recommended that the user provide the fragment length distribution parameters (--fragment-length-mean and --fragment-length-sd). For paired-end data, RSEM will automatically learn a fragment length distribution from the data.
581 Please note that some of the default values for the Bowtie parameters are not the same as those defined for Bowtie itself.
583 The temporary directory and all intermediate files will be removed when RSEM finishes unless '--keep-intermediate-files' is specified.
585 With the "--calc-ci" option, 95% credibility intervals and posterior mean estimates will be calculated in addition to maximum likelihood estimates.
591 =item B<sample_name.genes.results>
593 File containing gene level expression estimates. The format of each
594 line in this file is:
596 gene_id expected_counts tau_value [pmc_value tau_pme_value tau_ci_lower_bound tau_ci_upper_bound] transcript_id_list
598 Fields are separated by the tab character. Fields within "[]" are only
599 presented if '--calc-ci' is set. pme stands for posterior mean
600 estimation. pmc stands for posterior mean counts. ci_lower_bound(l)
601 means the lower bound of the credibility intervals, ci_upper_bound(u)
602 means the upper bound of the credibility intervals. So the credibility
603 interval is [l, u]. 'transcript_id_list' is a space-separated list of
604 transcript_ids belonging to the gene.
606 =item B<sample_name.isoforms.results>
608 File containing isoform level expression values. The format of each
609 line in this file is:
611 transcript_id expected_counts tau_value [pmc_value tau_pme_value tau_ci_lower_bound tau_ci_upper_bound] other_attributes
613 Fields are separated by the tab character. 'other_attributes' are all
614 other attributes after attribute 'transcript_id' field in the GTF
615 file. If no other attributes are given or no GTF file is provided in
616 'rsem-prepare-reference', there will be no tab after the
619 =item B<sample_name.bam, sample_name.sorted.bam and sample_name.sorted.bam.bai>
621 Only generated when --out-bam is specified.
623 'sample_name.bam' is a BAM-formatted file of read alignments in
624 genomic coordinates. Alignments of reads that have identical genomic
625 coordinates (i.e., alignments to different isoforms that share the
626 same genomic region) are collapsed into one alignment. The MAPQ field
627 of each alignment is set to min(100, floor(-10 * log10(1.0 - w) +
628 0.5)), where w is the posterior probability of that alignment being
629 the true mapping of a read. In addition, RSEM pads a new tag
630 ZW:f:value, where value is a single precision floating number
631 representing the posterior probability.
633 'sample_name.sorted.bam' and 'sample_name.sorted.bam.bai' are the
634 sorted BAM file and indices generated by samtools (included in RSEM package).
636 =item B<sample_name.stat>
638 This is a folder instead of a file. All model related statistics are stored in this folder. Use 'rsem-plot-model' can generate plots using this folder.
644 Assume the path to the bowtie executables is in the user's PATH environment variable. Reference files are under '/ref' with name 'mm9'.
646 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:
648 rsem-calculate-expression --phred64-quals \
655 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:
657 rsem-calculate-expression -p 8 \
662 mmliver_paired_end_quals
664 3) '/data/mmliver.fa', single-end reads without quality scores. We want to use 8 threads and generate a BAM file:
666 rsem-calculate-expression -p 8 \
670 mmliver_single_without_quals
672 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.
674 rsem-calculate-expression --bowtie-path /sw/bowtie \
676 --fragment-length-mean 150.0 \
677 --fragment-length-sd 35.0 \
686 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:
688 rsem-calculate-expression --paired-end \
691 /data/mmliver_paired_end_quals.bam \
693 mmliver_paired_end_quals