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 $strand_specific = 0;
58 GetOptions("keep-intermediate-files" => \$keep_intermediate_files,
59 "no-qualities" => \$no_qual,
60 "paired-end" => \$paired_end,
61 "strand-specific" => \$strand_specific,
64 "sam-header-info=s" => \$fn_list,
66 "seed-length=i" => \$L,
67 "bowtie-path=s" => \$bowtie_path,
70 "bowtie-m=i" => \$maxHits,
71 "phred33-quals" => \$phred33,
72 "phred64-quals" => \$phred64, #solexa1.3-quals" => \$phred64,
73 "solexa-quals" => \$solexa,
74 "forward-prob=f" => \$probF,
75 "fragment-length-min=i" => \$minL,
76 "fragment-length-max=i" => \$maxL,
77 "fragment-length-mean=f" => \$mean,
78 "fragment-length-sd=f" => \$sd,
79 "estimate-rspd" => \$estRSPD,
80 "num-rspd-bins=i" => \$B,
81 "p|num-threads=i" => \$nThreads,
82 "out-bam" => \$genBamF,
83 "calc-ci" => \$calcCI,
84 "ci-memory=i" => \$NMB,
86 "h|help" => \$help) or pod2usage(-exitval => 2, -verbose => 2);
88 pod2usage(-verbose => 2) if ($help == 1);
91 #check parameters and options
93 if ($is_sam || $is_bam) {
94 pod2usage(-msg => "Invalid number of arguments!", -exitval => 2, -verbose => 2) if (scalar(@ARGV) != 3);
95 pod2usage(-msg => "--sam and --bam cannot be active at the same time!", -exitval => 2, -verbose => 2) if ($is_sam == 1&& $is_bam == 1);
96 pod2usage(-msg => "--bowtie-path, --bowtie-n, --bowtie-e, --bowtie-m, --phred33-quals, --phred64-quals or --solexa-quals cannot be set if input is SAM/BAM format!", -exitval => 2, -verbose => 2) if ($bowtie_path ne "" || $C != 2 || $E != 99999999 || $maxHits != 200 || $phred33 || $phred64 || $solexa);
99 pod2usage(-msg => "Invalid number of arguments!", -exitval => 2, -verbose => 2) if (!$paired_end && scalar(@ARGV) != 3 || $paired_end && scalar(@ARGV) != 4);
100 pod2usage(-msg => "Only one of --phred33-quals --phred64-quals/--solexa1.3-quals --solexa-suqls can be active!", -exitval => 2, -verbose => 2) if ($phred33 + $phred64 + $solexa > 1);
101 podwusage(-msg => "--sam , --bam or --sam-header-info cannot be set if use bowtie aligner to produce alignments!", -exitval => 2, -verbose => 2) if ($is_sam || $is_bam || $fn_list ne "");
104 pod2usage(-msg => "Forward probability should be in [0, 1]!", -exitval => 2, -verbose => 2) if ($probF < 0 || $probF > 1);
105 pod2usage(-msg => "Min fragment length should be at least 1!", -exitval => 2, -verbose => 2) if ($minL < 1);
106 pod2usage(-msg => "Min fragment length should be smaller or equal to max fragment length!", -exitval => 2, -verbose => 2) if ($minL > $maxL);
107 pod2usage(-msg => "The memory allocated for calculating credibility intervals should be at least 1 MB!\n", -exitval => 2, -verbose => 2) if ($NMB < 1);
108 pod2usage(-msg => "Number of threads should be at least 1!\n", -exitval => 2, -verbose => 2) if ($nThreads < 1);
110 if ($strand_specific) { $probF = 1.0; }
116 my ($refName, $sampleName, $sampleToken, $temp_dir, $stat_dir, $imdName) = ();
120 if ($no_qual) { $read_type = 2; }
121 else { $read_type = 3; }
124 if ($no_qual) { $read_type = 0; }
125 else { $read_type = 1; }
128 if (scalar(@ARGV) == 3) {
129 if ($is_sam || $is_bam) { $inpF = $ARGV[0]; }
130 else {$mate1_list = $ARGV[0]; }
132 $sampleName = $ARGV[2];
135 $mate1_list = $ARGV[0];
136 $mate2_list = $ARGV[1];
138 $sampleName = $ARGV[3];
141 my $pos = rindex($sampleName, '/');
142 if ($pos < 0) { $sampleToken = $sampleName; }
143 else { $sampleToken = substr($sampleName, $pos + 1); }
145 $temp_dir = "$sampleName.temp";
146 $stat_dir = "$sampleName.stat";
148 if (!(-d $temp_dir) && !mkdir($temp_dir)) { print "Fail to create folder $temp_dir.\n"; exit(-1); }
149 if (!(-d $stat_dir) && !mkdir($stat_dir)) { print "Fail to create folder $stat_dir.\n"; exit(-1); }
151 $imdName = "$temp_dir/$sampleToken";
153 if (!$is_sam && !$is_bam && $phred33 + $phred64 + $solexa == 0) { $phred33 = 1; }
155 my ($mate_minL, $mate_maxL) = (1, $maxL);
157 if ($bowtie_path ne "") { $bowtie_path .= "/"; }
159 my ($fn, $dir, $suf) = fileparse($0);
162 if (!$is_sam && !$is_bam) {
163 $command = $bowtie_path."bowtie";
164 if ($read_type == 0 || $read_type == 2) { $command .= " -f"; }
165 else { $command .= " -q"; }
167 if ($phred33) { $command .= " --phred33-quals"; }
168 elsif ($phred64) { $command .= " --phred64-quals"; }
169 elsif ($solexa) { $command .= " --solexa-quals"; }
170 else { print "Oh, no!!!"; exit(2); }
172 $command .= " -n $C -e $E -l $L";
174 if ($read_type == 2 || $read_type == 3) { $command .= " -I $minL -X $maxL"; }
176 if ($strand_specific || $probF == 1.0) { $command .= " --norc"; }
177 elsif ($probF == 0.0) { $command .= " --nofw"; }
179 $command .= " -p $nThreads -a -m $maxHits -S";
180 if ($quiet) { $command .= " --quiet"; }
182 $command .= " $refName";
183 if ($read_type == 0 || $read_type == 1) {
184 $command .= " $mate1_list";
187 $command .= " -1 $mate1_list -2 $mate2_list";
190 $command .= " | gzip > $imdName.sam.gz";
192 $status = system($command);
194 print "bowtie failed! Please check if you provide correct parameters/options for the pipeline!\n";
199 $inpF = "$imdName.sam.gz";
200 $is_sam = 1; # output of bowtie is a sam file
203 $command = $dir."rsem-parse-alignments $refName $sampleName $sampleToken";
206 if ($is_sam) { $samInpType = "s"; }
207 elsif ($is_bam) { $samInpType = "b"; }
209 $command .= " $samInpType $inpF -t $read_type";
210 if ($fn_list ne "") { $command .= " -l $fn_list"; }
211 if ($tagName ne "") { $command .= " -tag $tagName"; }
212 if ($quiet) { $command .= " -q"; }
215 $status = system($command);
217 print "rsem-parse-alignments failed! Please check if you provide correct parameters/options for the pipeline!\n";
222 $command = $dir."rsem-build-read-index $gap";
224 case 0 { $command .= " 0 $quiet $imdName\_alignable.fa"; }
225 case 1 { $command .= " 1 $quiet $imdName\_alignable.fq"; }
226 case 2 { $command .= " 0 $quiet $imdName\_alignable_1.fa $imdName\_alignable_2.fa"; }
227 case 3 { $command .= " 1 $quiet $imdName\_alignable_1.fq $imdName\_alignable_2.fq"; }
230 $status = system($command);
232 print "rsem-build-read-index failed! Please check if you provide correct parameters/options for the pipeline!\n";
237 $status = open(OUTPUT, ">$imdName.mparams");
238 if ($status == 0) { print "Cannot generate $imdName.mparams!\n"; exit(-1); }
239 print OUTPUT "$minL $maxL\n";
240 print OUTPUT "$probF\n";
241 print OUTPUT "$estRSPD\n";
243 print OUTPUT "$mate_minL $mate_maxL\n";
244 print OUTPUT "$mean $sd\n";
248 $command = $dir."rsem-run-em $refName $read_type $sampleName $sampleToken -p $nThreads";
250 $command .= " -b $samInpType $inpF";
251 if ($fn_list ne "") { $command .= " 1 $fn_list"; }
252 else { $command .= " 0"; }
254 if ($calcCI) { $command .= " --gibbs-out"; }
255 if ($quiet) { $command .= " -q"; }
258 $status = system($command);
260 print "rsem-run-em failed! Please check if you provide correct parameters/options for the pipeline!\n";
266 $command = $dir."sam/samtools sort $sampleName.bam $sampleName.sorted";
268 $status = system($command);
270 print "sam/samtools sort failed! Please check if you provide correct parameters/options for the pipeline!\n";
274 $command = $dir."sam/samtools index $sampleName.sorted.bam";
276 $status = system($command);
278 print "sam/samtools index failed! Please check if you provide correct parameters/options for the pipeline!\n";
284 &collectResults("$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
285 &collectResults("$imdName.gene_res", "$sampleName.genes.results"); # gene level
288 $command = $dir."rsem-run-gibbs $refName $sampleName $sampleToken $BURNIN $CHAINLEN $SAMPLEGAP";
289 if ($quiet) { $command .= " -q"; }
291 $status = system($command);
293 print "rsem-run-gibbs failed! Please check if you provide correct parameters/options for the pipeline!\n";
298 system("mv $sampleName.isoforms.results $imdName.isoforms.results.bak1");
299 system("mv $sampleName.genes.results $imdName.genes.results.bak1");
300 &collectResults("$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
301 &collectResults("$imdName.gene_res", "$sampleName.genes.results"); # gene level
303 $command = $dir."rsem-calculate-credibility-intervals $refName $sampleName $sampleToken $CONFIDENCE $NSPC $NMB";
304 if ($quiet) { $command .= " -q"; }
306 $status = system($command);
308 print "rsem-calculate-credibility-intervals failed! Please check if you provide correct parameters/options for the pipeline!\n";
313 system("mv $sampleName.isoforms.results $imdName.isoforms.results.bak2");
314 system("mv $sampleName.genes.results $imdName.genes.results.bak2");
315 &collectResults("$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
316 &collectResults("$imdName.gene_res", "$sampleName.genes.results"); # gene level
319 if (!$keep_intermediate_files) {
320 $status = system ("rm -rf $tmp_dir");
322 print "Fail to delete the temporary folder!\n";
331 my (@results, @comment) = ();
338 $local_status = open(INPUT, $inpF);
339 if ($local_status == 0) { print "Fail to open file $inpF!\n"; exit(-1); }
344 while ($line = <INPUT>) {
347 my @local_arr = split(/\t/, $line);
348 if ($cnt == 4) { @comment = @local_arr; }
349 else { push(@results, \@local_arr); }
352 push(@results, \@comment);
355 $local_status = open(OUTPUT, ">$outF");
356 if ($local_status == 0) { print "Fail to create file $outF!\n"; exit(-1); }
358 my $n = scalar(@results);
359 my $m = scalar(@{$results[0]});
360 for (my $i = 0; $i < $m; $i++) {
362 for (my $j = 0; $j < $n; $j++) { push(@out_arr, $results[$j][$i]); }
364 print OUTPUT "@out_arr\n";
374 rsem-calculate-expression
380 rsem-calculate-expression [options] upstream_read_file(s) reference_name sample_name
381 rsem-calculate-expression [options] --paired-end upstream_read_file(s) downstream_read_file(s) reference_name sample_name
382 rsem-calculate-expression [options] --sam/--bam [--paired-end] input reference_name sample_name
390 =item B<upstream_read_files(s)>
392 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.
394 =item B<downstream_read_file(s)>
396 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.
400 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.
402 =item B<reference_name>
404 The name of the reference used. The user must have run 'rsem-prepare-reference' with this reference_name before running this program.
408 The name of the sample analyzed. All output files are prefixed by this name (e.g., sample_name.genes.results)
416 =item B<--paired-end>
418 Input reads are paired-end reads. (Default: off)
420 =item B<--no-qualities>
422 Input reads do not contain quality scores. (Default: off)
424 =item B<--strand-specific>
426 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)
430 Input file is in SAM format. (Default: off)
434 Input file is in BAM format. (Default: off)
436 =item B<--sam-header-info> <file>
438 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: "")
440 =item B<-p/--num-threads> <int>
442 Number of threads to use. Both Bowtie and expression estimation will use this many threads. (Default: 1)
446 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)
450 Calculate 95% credibility intervals and posterior mean estimates. (Default: off)
452 =item B<--seed-length> <int>
454 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)
456 =item B<--tag> <string>
458 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: "")
460 =item B<--bowtie-path> <path>
462 The path to the bowtie executables. (Default: the path to the bowtie executables is assumed to be in the user's PATH environment variable)
464 =item B<--bowtie-n> <int>
466 (Bowtie parameter) max # of mismatches in the seed. (Range: 0-3, Default: 2)
468 =item B<--bowtie-e> <int>
470 (Bowtie parameter) max sum of mismatch quality scores across the alignment. (Default: 99999999)
472 =item B<--bowtie-m> <int>
474 (Bowtie parameter) suppress all alignments for a read if > <int> valid alignments exist. (Default: 200)
476 =item B<--phred33-quals>
478 Input quality scores are encoded as Phred+33. (Default: on)
480 =item B<--phred64-quals>
482 Input quality scores are encoded as Phred+64 (default for GA Pipeline ver. >= 1.3). (Default: off)
484 =item B<--solexa-quals>
486 Input quality scores are solexa encoded (from GA Pipeline ver. < 1.3). (Default: off)
488 =item B<--forward-prob> <double>
490 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)
492 =item B<--fragment-length-min> <int>
494 Minimum read/insert length allowed. This is also the value for the bowtie -I option. (Default: 1)
496 =item B<--fragment-length-max> <int>
498 Maximum read/insert length allowed. This is also the value for the bowtie -X option. (Default: 1000)
500 =item B<--fragment-length-mean> <double>
502 (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)
504 =item B<--fragment-length-sd> <double>
506 (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>)
508 =item B<--estimate-rspd>
510 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)
512 =item B<--num-rspd-bins> <int>
514 Number of bins in the RSPD. Only relevant when '--estimate-rspd' is specified. Use of the default setting is recommended. (Default: 20)
516 =item B<--ci-memory> <int>
518 Amount of memory (in MB) RSEM is allowed to use for computing credibility intervals. (Default: 1024)
520 =item B<--keep-intermediate-files>
522 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)
526 Suppress the output of logging information. (Default: off)
530 Show help information.
536 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.
538 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:
540 sort -k 1,1 -s input.sam > input.sorted.sam
542 The SAM/BAM format RSEM uses is v1.3. However, it is compatible with old SAM/BAM format.
544 The user must run 'rsem-prepare-reference' with the appropriate reference before using this program.
546 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.
548 Please note that some of the default values for the Bowtie parameters are not the same as those defined for Bowtie itself.
550 The temporary directory and all intermediate files will be removed when RSEM finishes unless '--keep-intermediate-files' is specified.
552 With the "--calc-ci" option, 95% credibility intervals and posterior mean estimates will be calculated in addition to maximum likelihood estimates.
558 =item B<sample_name.genes.results>
560 File containing gene level expression estimates. The format of each
561 line in this file is:
563 gene_id expected_counts tau_value [pmc_value tau_pme_value tau_ci_lower_bound tau_ci_upper_bound] transcript_id_list
565 Fields are separated by the tab character. Fields within "[]" are only
566 presented if '--calc-ci' is set. pme stands for posterior mean
567 estimation. pmc stands for posterior mean counts. ci_lower_bound(l)
568 means the lower bound of the credibility intervals, ci_upper_bound(u)
569 means the upper bound of the credibility intervals. So the credibility
570 interval is [l, u]. 'transcript_id_list' is a space-separated list of
571 transcript_ids belonging to the gene.
573 =item B<sample_name.isoforms.results>
575 File containing isoform level expression values. The format of each
576 line in this file is:
578 transcript_id expected_counts tau_value [pmc_value tau_pme_value tau_ci_lower_bound tau_ci_upper_bound] other_attributes
580 Fields are separated by the tab character. 'other_attributes' are all
581 other attributes after attribute 'transcript_id' field in the GTF
582 file. If no other attributes are given or no GTF file is provided in
583 'rsem-prepare-reference', there will be no tab after the
586 =item B<sample_name.bam, sample_name.sorted.bam and sample_name.sorted.bam.bai>
588 Only generated when --out-bam is specified.
590 'sample_name.bam' is a BAM-formatted file of read alignments in
591 genomic coordinates. Alignments of reads that have identical genomic
592 coordinates (i.e., alignments to different isoforms that share the
593 same genomic region) are collapsed into one alignment. The MAPQ field
594 of each alignment is set to max(100, floor(-10 * log10(1.0 - w) +
595 0.5)), where w is the posterior probability of that alignment being
596 the true mapping of a read. In addition, RSEM pads a new tag
597 ZW:f:value, where value is a single precision floating number
598 representing the posterior probability.
600 'sample_name.sorted.bam' and 'sample_name.sorted.bam.bai' are the
601 sorted BAM file and indices generated by samtools (included in RSEM package).
603 =item B<sample_name.stat>
605 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.
611 Assume the path to the bowtie executables is in the user's PATH environment variable. Reference files are under '/ref' with name 'mm9'.
613 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:
615 rsem-calculate-expression --phred64-quals \
622 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:
624 rsem-calculate-expression -p 8 \
629 mmliver_paired_end_quals
631 3) '/data/mmliver.fa', single-end reads without quality scores. We want to use 8 threads and generate a BAM file:
633 rsem-calculate-expression -p 8 \
637 mmliver_single_without_quals
639 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.
641 rsem-calculate-expression --bowtie-path /sw/bowtie \
643 --fragment-length-mean 150.0 \
644 --fragment-length-sd 35.0 \
653 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:
655 rsem-calculate-expression --paired-end \
658 /data/mmliver_paired_end_quals.bam \
660 mmliver_paired_end_quals