7 use lib $FindBin::RealBin;
8 use rsem_perl_utils qw(runCommand collectResults showVersionInfo);
11 push(@PATH, $FindBin::RealBin, "$FindBin::RealBin/sam");
19 my $CONFIDENCE = 0.95;
22 my $NMB = 1024; # default
23 my $SortMem = "1G"; # default as 1G per thread
27 my $read_type = 1; # default, single end with qual
34 my $chunkMbs = 0; # 0 = use bowtie default
55 my $genBamF = 1; # default is generating transcript bam file
56 my $genGenomeBamF = 0;
60 my $var_opt = 0; # temporarily, only for internal use
66 my $keep_intermediate_files = 0;
68 my $strand_specific = 0;
71 my $bowtie2_path = "";
72 my $bowtie2_mismatch_rate = 0.1;
74 my $bowtie2_sensitivity_level = "sensitive"; # must be one of "very_fast", "fast", "sensitive", "very_sensitive"
79 my ($time_start, $time_end, $time_alignment, $time_rsem, $time_ci) = (0, 0, 0, 0, 0);
85 my ($refName, $sampleName, $sampleToken, $temp_dir, $stat_dir, $imdName, $statName) = ();
90 GetOptions("keep-intermediate-files" => \$keep_intermediate_files,
91 "temporary-folder=s" => \$temp_dir,
92 "no-qualities" => \$no_qual,
93 "paired-end" => \$paired_end,
94 "strand-specific" => \$strand_specific,
97 "sam-header-info=s" => \$fn_list,
99 "seed-length=i" => \$L,
100 "bowtie-path=s" => \$bowtie_path,
103 "bowtie-m=i" => \$maxHits,
104 "bowtie-chunkmbs=i" => \$chunkMbs,
105 "phred33-quals" => \$phred33,
106 "phred64-quals" => \$phred64, #solexa1.3-quals" => \$phred64,
107 "solexa-quals" => \$solexa,
108 "bowtie2" => \$bowtie2,
109 "bowtie2-path=s" => \$bowtie2_path,
110 "bowtie2-mismatch-rate=f" => \$bowtie2_mismatch_rate,
111 "bowtie2-k=i" => \$bowtie2_k,
112 "bowtie2-sensitivity-level=s" => \$bowtie2_sensitivity_level,
113 "forward-prob=f" => \$probF,
114 "fragment-length-min=i" => \$minL,
115 "fragment-length-max=i" => \$maxL,
116 "fragment-length-mean=f" => \$mean,
117 "fragment-length-sd=f" => \$sd,
118 "estimate-rspd" => \$estRSPD,
119 "num-rspd-bins=i" => \$B,
120 "p|num-threads=i" => \$nThreads,
121 "no-bam-output" => sub { $genBamF = 0; },
122 "output-genome-bam" => \$genGenomeBamF,
123 "sampling-for-bam" => \$sampling,
124 "calc-pme" => \$calcPME,
126 "calc-ci" => \$calcCI,
127 "ci-memory=i" => \$NMB,
128 "samtools-sort-mem=s" => \$SortMem,
130 "version" => \$version,
131 "q|quiet" => \$quiet,
132 "h|help" => \$help) or pod2usage(-exitval => 2, -verbose => 2);
134 pod2usage(-verbose => 2) if ($help == 1);
135 &showVersionInfo($FindBin::RealBin) if ($version == 1);
137 #check parameters and options
139 if ($is_sam || $is_bam) {
140 pod2usage(-msg => "Invalid number of arguments!", -exitval => 2, -verbose => 2) if (scalar(@ARGV) != 3);
141 pod2usage(-msg => "--sam and --bam cannot be active at the same time!", -exitval => 2, -verbose => 2) if ($is_sam == 1&& $is_bam == 1);
142 pod2usage(-msg => "--bowtie-path, --bowtie-n, --bowtie-e, --bowtie-m, --phred33-quals, --phred64-quals, --solexa-quals, --bowtie2, --bowtie2-path, --bowtie2-mismatch-rate, --bowtie2-k and --bowtie2-sensitivity-level cannot be set if input is SAM/BAM format!", -exitval => 2, -verbose => 2) if ($bowtie_path ne "" || $C != 2 || $E != 99999999 || $maxHits != 200 || $phred33 || $phred64 || $solexa || $bowtie2 || $bowtie2_path ne "" || $bowtie2_mismatch_rate != 0.1 || $bowtie2_k != 200 || $bowtie2_sensitivity_level ne "sensitive");
145 pod2usage(-msg => "Invalid number of arguments!", -exitval => 2, -verbose => 2) if (!$paired_end && scalar(@ARGV) != 3 || $paired_end && scalar(@ARGV) != 4);
146 pod2usage(-msg => "If --no-qualities is set, neither --phred33-quals, --phred64-quals or --solexa-quals can be active!", -exitval => 2, -verbose => 2) if ($no_qual && ($phred33 + $phred64 + $solexa > 0));
147 pod2usage(-msg => "Only one of --phred33-quals, --phred64-quals, and --solexa-quals can be active!", -exitval => 2, -verbose => 2) if ($phred33 + $phred64 + $solexa > 1);
148 pod2usage(-msg => "--sam , --bam or --sam-header-info cannot be set if use bowtie/bowtie2 aligner to produce alignments!", -exitval => 2, -verbose => 2) if ($is_sam || $is_bam || $fn_list ne "");
149 pod2usage(-msg => "--bowtie2-path, --bowtie2-mismatch-rate, --bowtie2-k and --bowtie2-sensitivity-level cannot be set if bowtie aligner is used!", -exitval => 2, -verbose => 2) if (!$bowtie2 && ($bowtie2_path ne "" || $bowtie2_mismatch_rate != 0.1 || $bowtie2_k != 200 || $bowtie2_sensitivity_level ne "sensitive"));
150 pod2usage(-msg => "--bowtie-path, --bowtie-n, --bowtie-e, --bowtie-m cannot be set if bowtie2 aligner is used!", -exitval => 2, -verbose => 2) if ($bowtie2 && ($bowtie_path ne "" || $C != 2 || $E != 99999999 || $maxHits != 200));
151 pod2usage(-msg => "Mismatch rate must be within [0, 1]!", -exitval => 2, -verbose => 2) if ($bowtie2 && ($bowtie2_mismatch_rate < 0.0 || $bowtie2_mismatch_rate > 1.0));
152 pod2usage(-msg => "Sensitivity level must be one of \"very_fast\", \"fast\", \"sensitive\", and \"very_sensitive\"!", -exitval => 2, -verbose => 2) if ($bowtie2 && (($bowtie2_sensitivity_level ne "very_fast") && ($bowtie2_sensitivity_level ne "fast") && ($bowtie2_sensitivity_level ne "sensitive") && ($bowtie2_sensitivity_level ne "very_sensitive")));
155 pod2usage(-msg => "Forward probability should be in [0, 1]!", -exitval => 2, -verbose => 2) if ($probF < 0 || $probF > 1);
156 pod2usage(-msg => "Min fragment length should be at least 1!", -exitval => 2, -verbose => 2) if ($minL < 1);
157 pod2usage(-msg => "Min fragment length should be smaller or equal to max fragment length!", -exitval => 2, -verbose => 2) if ($minL > $maxL);
158 pod2usage(-msg => "The memory allocated for calculating credibility intervals should be at least 1 MB!\n", -exitval => 2, -verbose => 2) if ($NMB < 1);
159 pod2usage(-msg => "Number of threads should be at least 1!\n", -exitval => 2, -verbose => 2) if ($nThreads < 1);
160 pod2usage(-msg => "Seed length should be at least 5!\n", -exitval => 2, -verbose => 2) if ($L < 5);
161 pod2usage(-msg => "--sampling-for-bam cannot be specified if --no-bam-output is specified!\n", -exitval => 2, -verbose => 2) if ($sampling && !$genBamF);
162 pod2usage(-msg => "--output-genome-bam cannot be specified if --no-bam-output is specified!\n", -exitval => 2, -verbose => 2) if ($genGenomeBamF && !$genBamF);
164 if ($L < 25) { print "Warning: the seed length set is less than 25! This is only allowed if the references are not added poly(A) tails.\n"; }
166 if ($strand_specific) { $probF = 1.0; }
169 if ($no_qual) { $read_type = 2; }
170 else { $read_type = 3; }
173 if ($no_qual) { $read_type = 0; }
174 else { $read_type = 1; }
177 if (scalar(@ARGV) == 3) {
178 if ($is_sam || $is_bam) { $inpF = $ARGV[0]; }
179 else {$mate1_list = $ARGV[0]; }
181 $sampleName = $ARGV[2];
184 $mate1_list = $ARGV[0];
185 $mate2_list = $ARGV[1];
187 $sampleName = $ARGV[3];
190 if (((-e "$refName.ta") && !(-e "$refName.gt")) || (!(-e "$refName.ta") && (-e "$refName.gt"))) {
191 print "Allele-specific expression related reference files are corrupted!\n";
195 $alleleS = (-e "$refName.ta") && (-e "$refName.gt");
197 if ($genGenomeBamF) {
198 open(INPUT, "$refName.ti");
199 my $line = <INPUT>; chomp($line);
201 my ($M, $type) = split(/ /, $line);
202 pod2usage(-msg => "No genome information provided, so genome bam file cannot be generated!\n", -exitval => 2, -verbose => 2) if ($type != 0);
205 my $pos = rindex($sampleName, '/');
206 if ($pos < 0) { $sampleToken = $sampleName; }
207 else { $sampleToken = substr($sampleName, $pos + 1); }
209 if ($temp_dir eq "") { $temp_dir = "$sampleName.temp"; }
210 $stat_dir = "$sampleName.stat";
212 if (!(-d $temp_dir) && !mkdir($temp_dir)) { print "Fail to create folder $temp_dir.\n"; exit(-1); }
213 if (!(-d $stat_dir) && !mkdir($stat_dir)) { print "Fail to create folder $stat_dir.\n"; exit(-1); }
215 $imdName = "$temp_dir/$sampleToken";
216 $statName = "$stat_dir/$sampleToken";
218 if (!$is_sam && !$is_bam && !$no_qual && ($phred33 + $phred64 + $solexa == 0)) { $phred33 = 1; }
220 my ($mate_minL, $mate_maxL) = (1, $maxL);
222 if ($bowtie_path ne "") { $bowtie_path .= "/"; }
223 if ($bowtie2_path ne "") { $bowtie2_path .= "/"; }
227 if (!$is_sam && !$is_bam) {
229 $command = $bowtie_path."bowtie";
230 if ($no_qual) { $command .= " -f"; }
231 else { $command .= " -q"; }
233 if ($phred33) { $command .= " --phred33-quals"; }
234 elsif ($phred64) { $command .= " --phred64-quals"; }
235 elsif ($solexa) { $command .= " --solexa-quals"; }
237 $command .= " -n $C -e $E -l $L";
238 if ($read_type == 2 || $read_type == 3) { $command .= " -I $minL -X $maxL"; }
239 if ($chunkMbs > 0) { $command .= " --chunkmbs $chunkMbs"; }
241 if ($strand_specific || $probF == 1.0) { $command .= " --norc"; }
242 elsif ($probF == 0.0) { $command .= " --nofw"; }
244 $command .= " -p $nThreads -a -m $maxHits -S";
245 if ($quiet) { $command .= " --quiet"; }
247 $command .= " $refName";
248 if ($read_type == 0 || $read_type == 1) {
249 $command .= " $mate1_list";
252 $command .= " -1 $mate1_list -2 $mate2_list";
255 # pipe to samtools to generate a BAM file
256 $command .= " | samtools view -S -b -o $imdName.bam -";
259 $command = $bowtie2_path."bowtie2";
260 if ($no_qual) { $command .= " -f"; }
261 else { $command .= " -q"; }
263 if ($phred33) { $command .= " --phred33"; }
264 elsif ($phred64) { $command .= " --phred64"; }
265 elsif ($solexa) { $command .= " --solexa-quals"; }
267 if ($bowtie2_sensitivity_level eq "very_fast") { $command .= " --very-fast"; }
268 elsif ($bowtie2_sensitivity_level eq "fast") { $command .= " --fast"; }
269 elsif ($bowtie2_sensitivity_level eq "sensitive") { $command .= " --sensitive"; }
270 else { $command .= " --very-sensitive"; }
272 $command .= " --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-$bowtie2_mismatch_rate";
274 if ($read_type == 2 || $read_type == 3) { $command .= " -I $minL -X $maxL --no-mixed --no-discordant"; }
276 if ($strand_specific || $probF == 1.0) { $command .= " --norc"; }
277 elsif ($probF == 0.0) { $command .= " --nofw"; }
279 $command .= " -p $nThreads -k $bowtie2_k";
280 if ($quiet) { $command .= " --quiet"; }
282 $command .= " -x $refName";
283 if ($read_type == 0 || $read_type == 1) {
284 $command .= " -U $mate1_list";
287 $command .= " -1 $mate1_list -2 $mate2_list";
290 # pipe to samtools to generate a BAM file
291 $command .= " | samtools view -S -b -o $imdName.bam -";
294 if ($mTime) { $time_start = time(); }
296 &runCommand($command);
298 if ($mTime) { $time_end = time(); $time_alignment = $time_end - $time_start; }
300 $inpF = "$imdName.bam";
301 $is_bam = 1; # alignments are outputed as a BAM file
304 if ($mTime) { $time_start = time(); }
306 $command = "rsem-parse-alignments $refName $imdName $statName";
309 if ($is_sam) { $samInpType = "s"; }
310 elsif ($is_bam) { $samInpType = "b"; }
312 $command .= " $samInpType $inpF -t $read_type";
313 if ($fn_list ne "") { $command .= " -l $fn_list"; }
314 if ($tagName ne "") { $command .= " -tag $tagName"; }
315 if ($quiet) { $command .= " -q"; }
317 &runCommand($command);
319 $command = "rsem-build-read-index $gap";
320 if ($read_type == 0) { $command .= " 0 $quiet $imdName\_alignable.fa"; }
321 elsif ($read_type == 1) { $command .= " 1 $quiet $imdName\_alignable.fq"; }
322 elsif ($read_type == 2) { $command .= " 0 $quiet $imdName\_alignable_1.fa $imdName\_alignable_2.fa"; }
323 elsif ($read_type == 3) { $command .= " 1 $quiet $imdName\_alignable_1.fq $imdName\_alignable_2.fq"; }
324 else { print "Impossible! read_type is not in [1,2,3,4]!\n"; exit(-1); }
325 &runCommand($command);
327 my $doesOpen = open(OUTPUT, ">$imdName.mparams");
328 if ($doesOpen == 0) { print "Cannot generate $imdName.mparams!\n"; exit(-1); }
329 print OUTPUT "$minL $maxL\n";
330 print OUTPUT "$probF\n";
331 print OUTPUT "$estRSPD\n";
333 print OUTPUT "$mate_minL $mate_maxL\n";
334 print OUTPUT "$mean $sd\n";
338 $command = "rsem-run-em $refName $read_type $sampleName $imdName $statName -p $nThreads";
340 $command .= " -b $samInpType $inpF";
341 if ($fn_list ne "") { $command .= " 1 $fn_list"; }
342 else { $command .= " 0"; }
343 if ($sampling) { $command .= " --sampling"; }
345 if ($calcPME || $var_opt || $calcCI) { $command .= " --gibbs-out"; }
346 if ($quiet) { $command .= " -q"; }
348 &runCommand($command);
351 &collectResults("allele", "$imdName.allele_res", "$sampleName.alleles.results"); # allele level
352 &collectResults("isoform", "$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
353 &collectResults("gene", "$imdName.gene_res", "$sampleName.genes.results"); # gene level
356 &collectResults("isoform", "$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
357 &collectResults("gene", "$imdName.gene_res", "$sampleName.genes.results"); # gene level
361 $command = "samtools sort -@ $nThreads -m $SortMem $sampleName.transcript.bam $sampleName.transcript.sorted";
362 &runCommand($command);
363 $command = "samtools index $sampleName.transcript.sorted.bam";
364 &runCommand($command);
366 if ($genGenomeBamF) {
367 $command = "rsem-tbam2gbam $refName $sampleName.transcript.bam $sampleName.genome.bam";
368 &runCommand($command);
369 $command = "samtools sort -@ $nThreads -m $SortMem $sampleName.genome.bam $sampleName.genome.sorted";
370 &runCommand($command);
371 $command = "samtools index $sampleName.genome.sorted.bam";
372 &runCommand($command);
376 if ($mTime) { $time_end = time(); $time_rsem = $time_end - $time_start; }
378 if ($mTime) { $time_start = time(); }
380 if ($calcPME || $var_opt || $calcCI ) {
381 $command = "rsem-run-gibbs $refName $imdName $statName $BURNIN $NCV $SAMPLEGAP";
382 $command .= " -p $nThreads";
383 if ($var_opt) { $command .= " --var"; }
384 if ($quiet) { $command .= " -q"; }
385 &runCommand($command);
388 if ($calcPME || $calcCI) {
390 system("mv $sampleName.alleles.results $imdName.alleles.results.bak1");
391 system("mv $sampleName.isoforms.results $imdName.isoforms.results.bak1");
392 system("mv $sampleName.genes.results $imdName.genes.results.bak1");
393 &collectResults("allele", "$imdName.allele_res", "$sampleName.alleles.results"); # allele level
394 &collectResults("isoform", "$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
395 &collectResults("gene", "$imdName.gene_res", "$sampleName.genes.results"); # gene level
398 system("mv $sampleName.isoforms.results $imdName.isoforms.results.bak1");
399 system("mv $sampleName.genes.results $imdName.genes.results.bak1");
400 &collectResults("isoform", "$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
401 &collectResults("gene", "$imdName.gene_res", "$sampleName.genes.results"); # gene level
406 $command = "rsem-calculate-credibility-intervals $refName $imdName $statName $CONFIDENCE $NCV $NSPC $NMB";
407 $command .= " -p $nThreads";
408 if ($quiet) { $command .= " -q"; }
409 &runCommand($command);
412 system("mv $sampleName.alleles.results $imdName.alleles.results.bak2");
413 system("mv $sampleName.isoforms.results $imdName.isoforms.results.bak2");
414 system("mv $sampleName.genes.results $imdName.genes.results.bak2");
415 &collectResults("allele", "$imdName.allele_res", "$sampleName.alleles.results"); # allele level
416 &collectResults("isoform", "$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
417 &collectResults("gene", "$imdName.gene_res", "$sampleName.genes.results"); # gene level
420 system("mv $sampleName.isoforms.results $imdName.isoforms.results.bak2");
421 system("mv $sampleName.genes.results $imdName.genes.results.bak2");
422 &collectResults("isoform", "$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
423 &collectResults("gene", "$imdName.gene_res", "$sampleName.genes.results"); # gene level
427 if ($mTime) { $time_end = time(); $time_ci = $time_end - $time_start; }
429 if ($mTime) { $time_start = time(); }
431 if (!$keep_intermediate_files) {
432 &runCommand("rm -rf $temp_dir", "Fail to delete the temporary folder!");
435 if ($mTime) { $time_end = time(); }
438 open(OUTPUT, ">$sampleName.time");
439 print OUTPUT "Aligning reads: $time_alignment s.\n";
440 print OUTPUT "Estimating expression levels: $time_rsem s.\n";
441 print OUTPUT "Calculating credibility intervals: $time_ci s.\n";
442 # my $time_del = $time_end - $time_start;
443 # print OUTPUT "Delete: $time_del s.\n";
451 rsem-calculate-expression
455 rsem-calculate-expression [options] upstream_read_file(s) reference_name sample_name
456 rsem-calculate-expression [options] --paired-end upstream_read_file(s) downstream_read_file(s) reference_name sample_name
457 rsem-calculate-expression [options] --sam/--bam [--paired-end] input reference_name sample_name
463 =item B<upstream_read_files(s)>
465 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.
467 =item B<downstream_read_file(s)>
469 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.
473 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.
475 =item B<reference_name>
477 The name of the reference used. The user must have run 'rsem-prepare-reference' with this reference_name before running this program.
481 The name of the sample analyzed. All output files are prefixed by this name (e.g., sample_name.genes.results)
489 =item B<--paired-end>
491 Input reads are paired-end reads. (Default: off)
493 =item B<--no-qualities>
495 Input reads do not contain quality scores. (Default: off)
497 =item B<--strand-specific>
499 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/Bowtie 2 aligner, the '--norc' Bowtie/Bowtie 2 option will be used, which disables alignment to the reverse strand of transcripts. (Default: off)
503 Input file is in SAM format. (Default: off)
507 Input file is in BAM format. (Default: off)
509 =item B<--sam-header-info> <file>
511 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: "")
513 =item B<-p/--num-threads> <int>
515 Number of threads to use. Both Bowtie/Bowtie2, expression estimation and 'samtools sort' will use this many threads. (Default: 1)
517 =item B<--no-bam-output>
519 Do not output any BAM file. (Default: off)
521 =item B<--output-genome-bam>
523 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)
525 =item B<--sampling-for-bam>
527 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)
531 Run RSEM's collapsed Gibbs sampler to calculate posterior mean estimates. (Default: off)
535 Calculate 95% credibility intervals and posterior mean estimates. (Default: off)
537 =item B<--seed-length> <int>
539 Seed length used by the read aligner. Providing the correct value is important for RSEM. If RSEM runs Bowtie, it uses this value for Bowtie's seed length parameter. Any read with its or at least one of its mates' (for paired-end reads) length less than this value will be ignored. If the references are not added poly(A) tails, the minimum allowed value is 5, otherwise, the minimum allowed value is 25. Note that this script will only check if the value >= 5 and give a warning message if the value < 25 but >= 5. (Default: 25)
541 =item B<--tag> <string>
543 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: "")
545 =item B<--bowtie-path> <path>
547 The path to the Bowtie executables. (Default: the path to the Bowtie executables is assumed to be in the user's PATH environment variable)
549 =item B<--bowtie-n> <int>
551 (Bowtie parameter) max # of mismatches in the seed. (Range: 0-3, Default: 2)
553 =item B<--bowtie-e> <int>
555 (Bowtie parameter) max sum of mismatch quality scores across the alignment. (Default: 99999999)
557 =item B<--bowtie-m> <int>
559 (Bowtie parameter) suppress all alignments for a read if > <int> valid alignments exist. (Default: 200)
561 =item B<--bowtie-chunkmbs> <int>
563 (Bowtie parameter) memory allocated for best first alignment calculation (Default: 0 - use Bowtie's default)
565 =item B<--phred33-quals>
567 Input quality scores are encoded as Phred+33. (Default: on)
569 =item B<--phred64-quals>
571 Input quality scores are encoded as Phred+64 (default for GA Pipeline ver. >= 1.3). (Default: off)
573 =item B<--solexa-quals>
575 Input quality scores are solexa encoded (from GA Pipeline ver. < 1.3). (Default: off)
579 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)
581 =item B<--bowtie2-path> <path>
583 (Bowtie 2 parameter) The path to the Bowtie 2 executables. (Default: the path to the Bowtie 2 executables is assumed to be in the user's PATH environment variable)
585 =item B<--bowtie2-mismatch-rate> <double>
587 (Bowtie 2 parameter) The maximum mismatch rate allowed. (Default: 0.1)
589 =item B<--bowtie2-k> <int>
591 (Bowtie 2 parameter) Find up to <int> alignments per read. (Default: 200)
593 =item B<--bowtie2-sensitivity-level> <string>
595 (Bowtie 2 parameter) Set Bowtie 2's preset options in --end-to-end mode. This option controls how hard Bowtie 2 tries to find alignments. <string> must be one of "very_fast", "fast", "sensitive" and "very_sensitive". The four candidates correspond to Bowtie 2's "--very-fast", "--fast", "--sensitive" and "--very-sensitive" options. (Default: "sensitive" - use Bowtie 2's default)
597 =item B<--forward-prob> <double>
599 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)
601 =item B<--fragment-length-min> <int>
603 Minimum read/insert length allowed. This is also the value for the Bowtie/Bowtie2 -I option. (Default: 1)
605 =item B<--fragment-length-max> <int>
607 Maximum read/insert length allowed. This is also the value for the Bowtie/Bowtie 2 -X option. (Default: 1000)
609 =item B<--fragment-length-mean> <double>
611 (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)
613 =item B<--fragment-length-sd> <double>
615 (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>)
617 =item B<--estimate-rspd>
619 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)
621 =item B<--num-rspd-bins> <int>
623 Number of bins in the RSPD. Only relevant when '--estimate-rspd' is specified. Use of the default setting is recommended. (Default: 20)
625 =item B<--ci-memory> <int>
627 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)
629 =item B<--samtools-sort-mem> <string>
631 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)
633 =item B<--keep-intermediate-files>
635 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)
637 =item B<--temporary-folder> <string>
639 Set where to put the temporary files generated by RSEM. If the folder specified does not exist, RSEM will try to create it. (Default: sample_name.temp)
643 Output time consumed by each step of RSEM to 'sample_name.time'. (Default: off)
647 Suppress the output of logging information. (Default: off)
651 Show help information.
655 Show version information.
661 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.
663 One simple way to make the alignment file satisfying RSEM's requirements (assuming the aligner used put mates in a paired-end read adjacent) is to use 'convert-sam-for-rsem' script. This script only accept SAM format files as input. If a BAM format file is obtained, please use samtools to convert it to a SAM file first. For example, if '/ref/mouse_125' is the 'reference_name' and the SAM file is named 'input.sam', you can run the following command:
665 convert-sam-for-rsem /ref/mouse_125 input.sam -o input_for_rsem.sam
667 For details, please refer to 'convert-sam-for-rsem's documentation page.
669 The SAM/BAM format RSEM uses is v1.4. However, it is compatible with old SAM/BAM format. However, RSEM cannot recognize 0x100 in the FLAG field. In addition, RSEM requires SEQ and QUAL are not '*'.
671 The user must run 'rsem-prepare-reference' with the appropriate reference before using this program.
673 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.
675 Please note that some of the default values for the Bowtie parameters are not the same as those defined for Bowtie itself.
677 The temporary directory and all intermediate files will be removed when RSEM finishes unless '--keep-intermediate-files' is specified.
679 With the '--calc-ci' option, 95% credibility intervals and posterior mean estimates will be calculated in addition to maximum likelihood estimates.
685 =item B<sample_name.isoforms.results>
687 File containing isoform level expression estimates. The first line
688 contains column names separated by the tab character. The format of
689 each line in the rest of this file is:
691 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]
693 Fields are separated by the tab character. Fields within "[]" are
694 optional. They will not be presented if neither '--calc-pme' nor
697 'transcript_id' is the transcript name of this transcript. 'gene_id'
698 is the gene name of the gene which this transcript belongs to (denote
699 this gene as its parent gene). If no gene information is provided,
700 'gene_id' and 'transcript_id' are the same.
702 'length' is this transcript's sequence length (poly(A) tail is not
703 counted). 'effective_length' counts only the positions that can
704 generate a valid fragment. If no poly(A) tail is added,
705 'effective_length' is equal to transcript length - mean fragment
706 length + 1. If one transcript's effective length is less than 1, this
707 transcript's both effective length and abundance estimates are set to
710 'expected_count' is the sum of the posterior probability of each read
711 comes from this transcript over all reads. Because 1) each read
712 aligning to this transcript has a probability of being generated from
713 background noise; 2) RSEM may filter some alignable low quality reads,
714 the sum of expected counts for all transcript are generally less than
715 the total number of reads aligned.
717 'TPM' stands for Transcripts Per Million. It is a relative measure of
718 transcript abundance. The sum of all transcripts' TPM is 1
719 million. 'FPKM' stands for Fragments Per Kilobase of transcript per
720 Million mapped reads. It is another relative measure of transcript
721 abundance. If we define l_bar be the mean transcript length in a
722 sample, which can be calculated as
724 l_bar = \sum_i TPM_i / 10^6 * effective_length_i (i goes through every transcript),
726 the following equation is hold:
728 FPKM_i = 10^3 / l_bar * TPM_i.
730 We can see that the sum of FPKM is not a constant across samples.
732 'IsoPct' stands for isoform percentage. It is the percentage of this
733 transcript's abandunce over its parent gene's abandunce. If its parent
734 gene has only one isoform or the gene information is not provided,
735 this field will be set to 100.
737 'pme_expected_count', 'pme_TPM', 'pme_FPKM' are posterior mean
738 estimates calculated by RSEM's Gibbs sampler. 'IsoPct_from_pme_TPM' is
739 the isoform percentage calculated from 'pme_TPM' values.
741 'TPM_ci_lower_bound', 'TPM_ci_upper_bound', 'FPKM_ci_lower_bound' and
742 'FPKM_ci_upper_bound' are lower(l) and upper(u) bounds of 95%
743 credibility intervals for TPM and FPKM values. The bounds are
744 inclusive (i.e. [l, u]).
746 =item B<sample_name.genes.results>
748 File containing gene level expression estimates. The first line
749 contains column names separated by the tab character. The format of
750 each line in the rest of this file is:
752 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]
754 Fields are separated by the tab character. Fields within "[]" are
755 optional. They will not be presented if neither '--calc-pme' nor
758 'transcript_id(s)' is a comma-separated list of transcript_ids
759 belonging to this gene. If no gene information is provided, 'gene_id'
760 and 'transcript_id(s)' are identical (the 'transcript_id').
762 A gene's 'length' and 'effective_length' are
763 defined as the weighted average of its transcripts' lengths and
764 effective lengths (weighted by 'IsoPct'). A gene's abundance estimates
765 are just the sum of its transcripts' abundance estimates.
767 =item B<sample_name.alleles.results>
769 Only generated when the RSEM references are built with allele-specific
772 This file contains allele level expression estimates for
773 allele-specific expression calculation. The first line
774 contains column names separated by the tab character. The format of
775 each line in the rest of this file is:
777 allele_id transcript_id gene_id length effective_length expected_count TPM FPKM AlleleIsoPct AlleleGenePct [pme_expected_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]
779 Fields are separated by the tab character. Fields within "[]" are
780 optional. They will not be presented if neither '--calc-pme' nor
783 'allele_id' is the allele-specific name of this allele-specific transcript.
785 'AlleleIsoPct' stands for allele-specific percentage on isoform
786 level. It is the percentage of this allele-specific transcript's
787 abundance over its parent transcript's abundance. If its parent
788 transcript has only one allele variant form, this field will be set to
791 'AlleleGenePct' stands for allele-specific percentage on gene
792 level. It is the percentage of this allele-specific transcript's
793 abundance over its parent gene's abundance.
795 'AlleleIsoPct_from_pme_TPM' and 'AlleleGenePct_from_pme_TPM' have
796 similar meanings. They are calculated based on posterior mean
799 Please note that if this file is present, the fields 'length' and
800 'effective_length' in 'sample_name.isoforms.results' should be
801 interpreted similarly as the corresponding definitions in
802 'sample_name.genes.results'.
804 =item B<sample_name.transcript.bam, sample_name.transcript.sorted.bam and sample_name.transcript.sorted.bam.bai>
806 Only generated when --no-bam-output is not specified.
808 'sample_name.transcript.bam' is a BAM-formatted file of read
809 alignments in transcript coordinates. The MAPQ field of each alignment
810 is set to min(100, floor(-10 * log10(1.0 - w) + 0.5)), where w is the
811 posterior probability of that alignment being the true mapping of a
812 read. In addition, RSEM pads a new tag ZW:f:value, where value is a
813 single precision floating number representing the posterior
814 probability. Because this file contains all alignment lines produced
815 by bowtie or user-specified aligners, it can also be used as a
816 replacement of the aligner generated BAM/SAM file. For paired-end
817 reads, if one mate has alignments but the other does not, this file
818 marks the alignable mate as "unmappable" (flag bit 0x4) and appends an
819 optional field "Z0:A:!".
821 'sample_name.transcript.sorted.bam' and
822 'sample_name.transcript.sorted.bam.bai' are the sorted BAM file and
823 indices generated by samtools (included in RSEM package).
825 =item B<sample_name.genome.bam, sample_name.genome.sorted.bam and sample_name.genome.sorted.bam.bai>
827 Only generated when --no-bam-output is not specified and --output-genome-bam is specified.
829 'sample_name.genome.bam' is a BAM-formatted file of read alignments in
830 genomic coordinates. Alignments of reads that have identical genomic
831 coordinates (i.e., alignments to different isoforms that share the
832 same genomic region) are collapsed into one alignment. The MAPQ field
833 of each alignment is set to min(100, floor(-10 * log10(1.0 - w) +
834 0.5)), where w is the posterior probability of that alignment being
835 the true mapping of a read. In addition, RSEM pads a new tag
836 ZW:f:value, where value is a single precision floating number
837 representing the posterior probability. If an alignment is spliced, a
838 XS:A:value tag is also added, where value is either '+' or '-'
839 indicating the strand of the transcript it aligns to.
841 'sample_name.genome.sorted.bam' and 'sample_name.genome.sorted.bam.bai' are the
842 sorted BAM file and indices generated by samtools (included in RSEM package).
844 =item B<sample_name.time>
846 Only generated when --time is specified.
848 It contains time (in seconds) consumed by aligning reads, estimating expression levels and calculating credibility intervals.
850 =item B<sample_name.stat>
852 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.
858 Assume the path to the bowtie executables is in the user's PATH environment variable. Reference files are under '/ref' with name 'mouse_125'.
860 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:
862 rsem-calculate-expression --phred64-quals \
864 --output-genome-bam \
869 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:
871 rsem-calculate-expression -p 8 \
876 mmliver_paired_end_quals
878 3) '/data/mmliver.fa', single-end reads without quality scores. We want to use 8 threads:
880 rsem-calculate-expression -p 8 \
884 mmliver_single_without_quals
886 4) Data are the same as 1). This time we assume the bowtie executables are under '/sw/bowtie'. 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:
888 rsem-calculate-expression --bowtie-path /sw/bowtie \
890 --fragment-length-mean 150.0 \
891 --fragment-length-sd 35.0 \
893 --output-genome-bam \
900 5) '/data/mmliver_paired_end_quals.bam', paired-end reads with quality scores. We want to use 8 threads:
902 rsem-calculate-expression --paired-end \
905 /data/mmliver_paired_end_quals.bam \
907 mmliver_paired_end_quals