12 my $CONFIDENCE = 0.95;
15 my $NMB = 1024; # default
19 my $read_type = 1; # default, single end with qual
21 my @transcript_title = ("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");
23 my @gene_title = ("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");
30 my $chunkMbs = 0; # 0 = use bowtie default
51 my $genBamF = 1; # default is generating transcript bam file
52 my $genGenomeBamF = 0;
55 my $var_opt = 0; # temporarily, only for internal use
61 my $keep_intermediate_files = 0;
63 my $strand_specific = 0;
68 my ($time_start, $time_end, $time_alignment, $time_rsem, $time_ci) = (0, 0, 0, 0, 0);
74 my ($refName, $sampleName, $sampleToken, $temp_dir, $stat_dir, $imdName, $statName) = ();
77 GetOptions("keep-intermediate-files" => \$keep_intermediate_files,
78 "temporary-folder=s" => \$temp_dir,
79 "no-qualities" => \$no_qual,
80 "paired-end" => \$paired_end,
81 "strand-specific" => \$strand_specific,
84 "sam-header-info=s" => \$fn_list,
86 "seed-length=i" => \$L,
87 "bowtie-path=s" => \$bowtie_path,
90 "bowtie-m=i" => \$maxHits,
91 "bowtie-chunkmbs=i" => \$chunkMbs,
92 "phred33-quals" => \$phred33,
93 "phred64-quals" => \$phred64, #solexa1.3-quals" => \$phred64,
94 "solexa-quals" => \$solexa,
95 "forward-prob=f" => \$probF,
96 "fragment-length-min=i" => \$minL,
97 "fragment-length-max=i" => \$maxL,
98 "fragment-length-mean=f" => \$mean,
99 "fragment-length-sd=f" => \$sd,
100 "estimate-rspd" => \$estRSPD,
101 "num-rspd-bins=i" => \$B,
102 "p|num-threads=i" => \$nThreads,
103 "no-bam-output" => sub { $genBamF = 0; },
104 "output-genome-bam" => \$genGenomeBamF,
105 "sampling-for-bam" => \$sampling,
107 "calc-ci" => \$calcCI,
108 "ci-memory=i" => \$NMB,
110 "version" => \$version,
111 "q|quiet" => \$quiet,
112 "h|help" => \$help) or pod2usage(-exitval => 2, -verbose => 2);
114 my ($fn, $dir, $suf) = fileparse($0);
116 pod2usage(-verbose => 2) if ($help == 1);
117 &showVersionInfo($dir) if ($version == 1);
119 #check parameters and options
121 if ($is_sam || $is_bam) {
122 pod2usage(-msg => "Invalid number of arguments!", -exitval => 2, -verbose => 2) if (scalar(@ARGV) != 3);
123 pod2usage(-msg => "--sam and --bam cannot be active at the same time!", -exitval => 2, -verbose => 2) if ($is_sam == 1&& $is_bam == 1);
124 pod2usage(-msg => "--bowtie-path, --bowtie-n, --bowtie-e, --bowtie-m, --phred33-quals, --phred64-quals or --solexa-quals cannot be set if input is SAM/BAM format!", -exitval => 2, -verbose => 2) if ($bowtie_path ne "" || $C != 2 || $E != 99999999 || $maxHits != 200 || $phred33 || $phred64 || $solexa);
127 pod2usage(-msg => "Invalid number of arguments!", -exitval => 2, -verbose => 2) if (!$paired_end && scalar(@ARGV) != 3 || $paired_end && scalar(@ARGV) != 4);
128 pod2usage(-msg => "Only one of --phred33-quals --phred64-quals/--solexa1.3-quals --solexa-suqls can be active!", -exitval => 2, -verbose => 2) if ($phred33 + $phred64 + $solexa > 1);
129 podwusage(-msg => "--sam , --bam or --sam-header-info cannot be set if use bowtie aligner to produce alignments!", -exitval => 2, -verbose => 2) if ($is_sam || $is_bam || $fn_list ne "");
132 pod2usage(-msg => "Forward probability should be in [0, 1]!", -exitval => 2, -verbose => 2) if ($probF < 0 || $probF > 1);
133 pod2usage(-msg => "Min fragment length should be at least 1!", -exitval => 2, -verbose => 2) if ($minL < 1);
134 pod2usage(-msg => "Min fragment length should be smaller or equal to max fragment length!", -exitval => 2, -verbose => 2) if ($minL > $maxL);
135 pod2usage(-msg => "The memory allocated for calculating credibility intervals should be at least 1 MB!\n", -exitval => 2, -verbose => 2) if ($NMB < 1);
136 pod2usage(-msg => "Number of threads should be at least 1!\n", -exitval => 2, -verbose => 2) if ($nThreads < 1);
137 pod2usage(-msg => "Seed length should be at least 5!\n", -exitval => 2, -verbose => 2) if ($L < 5);
138 pod2usage(-msg => "--sampling-for-bam cannot be specified if --no-bam-output is specified!\n", -exitval => 2, -verbose => 2) if ($sampling && !$genBamF);
139 pod2usage(-msg => "--output-genome-bam cannot be specified if --no-bam-output is specified!\n", -exitval => 2, -verbose => 2) if ($genGenomeBamF && !$genBamF);
141 if ($L < 25) { print "Warning: the seed length set is less than 25! This is only allowed if the references are not added poly(A) tails.\n"; }
143 if ($strand_specific) { $probF = 1.0; }
146 if ($no_qual) { $read_type = 2; }
147 else { $read_type = 3; }
150 if ($no_qual) { $read_type = 0; }
151 else { $read_type = 1; }
154 if (scalar(@ARGV) == 3) {
155 if ($is_sam || $is_bam) { $inpF = $ARGV[0]; }
156 else {$mate1_list = $ARGV[0]; }
158 $sampleName = $ARGV[2];
161 $mate1_list = $ARGV[0];
162 $mate2_list = $ARGV[1];
164 $sampleName = $ARGV[3];
167 if ($genGenomeBamF) {
168 open(INPUT, "$refName.ti");
169 my $line = <INPUT>; chomp($line);
171 my ($M, $type) = split(/ /, $line);
172 pod2usage(-msg => "No genome information provided, so genome bam file cannot be generated!\n", -exitval => 2, -verbose => 2) if ($type != 0);
175 my $pos = rindex($sampleName, '/');
176 if ($pos < 0) { $sampleToken = $sampleName; }
177 else { $sampleToken = substr($sampleName, $pos + 1); }
179 if ($temp_dir eq "") { $temp_dir = "$sampleName.temp"; }
180 $stat_dir = "$sampleName.stat";
182 if (!(-d $temp_dir) && !mkdir($temp_dir)) { print "Fail to create folder $temp_dir.\n"; exit(-1); }
183 if (!(-d $stat_dir) && !mkdir($stat_dir)) { print "Fail to create folder $stat_dir.\n"; exit(-1); }
185 $imdName = "$temp_dir/$sampleToken";
186 $statName = "$stat_dir/$sampleToken";
188 if (!$is_sam && !$is_bam && !$no_qual && ($phred33 + $phred64 + $solexa == 0)) { $phred33 = 1; }
190 my ($mate_minL, $mate_maxL) = (1, $maxL);
192 if ($bowtie_path ne "") { $bowtie_path .= "/"; }
196 if (!$is_sam && !$is_bam) {
197 $command = $bowtie_path."bowtie";
198 if ($no_qual) { $command .= " -f"; }
199 else { $command .= " -q"; }
201 if ($phred33) { $command .= " --phred33-quals"; }
202 elsif ($phred64) { $command .= " --phred64-quals"; }
203 elsif ($solexa) { $command .= " --solexa-quals"; }
205 $command .= " -n $C -e $E -l $L";
206 if ($read_type == 2 || $read_type == 3) { $command .= " -I $minL -X $maxL"; }
207 if ($chunkMbs > 0) { $command .= " --chunkmbs $chunkMbs"; }
209 if ($strand_specific || $probF == 1.0) { $command .= " --norc"; }
210 elsif ($probF == 0.0) { $command .= " --nofw"; }
212 $command .= " -p $nThreads -a -m $maxHits -S";
213 if ($quiet) { $command .= " --quiet"; }
215 $command .= " $refName";
216 if ($read_type == 0 || $read_type == 1) {
217 $command .= " $mate1_list";
220 $command .= " -1 $mate1_list -2 $mate2_list";
223 # pipe to samtools to generate a BAM file
224 $command .= " | $dir\sam/samtools view -S -b -o $imdName.bam -";
226 if ($mTime) { $time_start = time(); }
228 &runCommand($command);
230 if ($mTime) { $time_end = time(); $time_alignment = $time_end - $time_start; }
232 $inpF = "$imdName.bam";
233 $is_bam = 1; # alignments are outputed as a BAM file
236 if ($mTime) { $time_start = time(); }
238 $command = $dir."rsem-parse-alignments $refName $imdName $statName";
241 if ($is_sam) { $samInpType = "s"; }
242 elsif ($is_bam) { $samInpType = "b"; }
244 $command .= " $samInpType $inpF -t $read_type";
245 if ($fn_list ne "") { $command .= " -l $fn_list"; }
246 if ($tagName ne "") { $command .= " -tag $tagName"; }
247 if ($quiet) { $command .= " -q"; }
249 &runCommand($command);
251 $command = $dir."rsem-build-read-index $gap";
252 if ($read_type == 0) { $command .= " 0 $quiet $imdName\_alignable.fa"; }
253 elsif ($read_type == 1) { $command .= " 1 $quiet $imdName\_alignable.fq"; }
254 elsif ($read_type == 2) { $command .= " 0 $quiet $imdName\_alignable_1.fa $imdName\_alignable_2.fa"; }
255 elsif ($read_type == 3) { $command .= " 1 $quiet $imdName\_alignable_1.fq $imdName\_alignable_2.fq"; }
256 else { print "Impossible! read_type is not in [1,2,3,4]!\n"; exit(-1); }
257 &runCommand($command);
259 my $doesOpen = open(OUTPUT, ">$imdName.mparams");
260 if ($doesOpen == 0) { print "Cannot generate $imdName.mparams!\n"; exit(-1); }
261 print OUTPUT "$minL $maxL\n";
262 print OUTPUT "$probF\n";
263 print OUTPUT "$estRSPD\n";
265 print OUTPUT "$mate_minL $mate_maxL\n";
266 print OUTPUT "$mean $sd\n";
270 $command = $dir."rsem-run-em $refName $read_type $sampleName $imdName $statName -p $nThreads";
272 $command .= " -b $samInpType $inpF";
273 if ($fn_list ne "") { $command .= " 1 $fn_list"; }
274 else { $command .= " 0"; }
275 if ($sampling) { $command .= " --sampling"; }
277 if ($calcCI || $var_opt) { $command .= " --gibbs-out"; }
278 if ($quiet) { $command .= " -q"; }
280 &runCommand($command);
282 &collectResults("isoform", "$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
283 &collectResults("gene", "$imdName.gene_res", "$sampleName.genes.results"); # gene level
286 $command = $dir."sam/samtools sort $sampleName.transcript.bam $sampleName.transcript.sorted";
287 &runCommand($command);
288 $command = $dir."sam/samtools index $sampleName.transcript.sorted.bam";
289 &runCommand($command);
291 if ($genGenomeBamF) {
292 $command = $dir."rsem-tbam2gbam $refName $sampleName.transcript.bam $sampleName.genome.bam";
293 &runCommand($command);
294 $command = $dir."sam/samtools sort $sampleName.genome.bam $sampleName.genome.sorted";
295 &runCommand($command);
296 $command = $dir."sam/samtools index $sampleName.genome.sorted.bam";
297 &runCommand($command);
301 if ($mTime) { $time_end = time(); $time_rsem = $time_end - $time_start; }
303 if ($mTime) { $time_start = time(); }
305 if ($calcCI || $var_opt) {
306 $command = $dir."rsem-run-gibbs $refName $imdName $statName $BURNIN $NCV $SAMPLEGAP";
307 $command .= " -p $nThreads";
308 if ($var_opt) { $command .= " --var"; }
309 if ($quiet) { $command .= " -q"; }
310 &runCommand($command);
314 system("mv $sampleName.isoforms.results $imdName.isoforms.results.bak1");
315 system("mv $sampleName.genes.results $imdName.genes.results.bak1");
316 &collectResults("isoform", "$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
317 &collectResults("gene", "$imdName.gene_res", "$sampleName.genes.results"); # gene level
319 $command = $dir."rsem-calculate-credibility-intervals $refName $imdName $statName $CONFIDENCE $NCV $NSPC $NMB";
320 $command .= " -p $nThreads";
321 if ($quiet) { $command .= " -q"; }
322 &runCommand($command);
324 system("mv $sampleName.isoforms.results $imdName.isoforms.results.bak2");
325 system("mv $sampleName.genes.results $imdName.genes.results.bak2");
326 &collectResults("isoform", "$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
327 &collectResults("gene", "$imdName.gene_res", "$sampleName.genes.results"); # gene level
330 if ($mTime) { $time_end = time(); $time_ci = $time_end - $time_start; }
332 if ($mTime) { $time_start = time(); }
334 if (!$keep_intermediate_files) {
335 &runCommand("rm -rf $temp_dir", "Fail to delete the temporary folder!");
338 if ($mTime) { $time_end = time(); }
341 open(OUTPUT, ">$sampleName.time");
342 print OUTPUT "Aligning reads: $time_alignment s.\n";
343 print OUTPUT "Estimating expression levels: $time_rsem s.\n";
344 print OUTPUT "Calculating credibility intervals: $time_ci s.\n";
345 my $time_del = $time_end - $time_start;
346 # print OUTPUT "Delete: $time_del s.\n";
353 my $status = system($_[0]);
356 if (scalar(@_) > 1) { $errmsg .= $_[1]."\n"; }
357 $errmsg .= "\"$_[0]\" failed! Please check if you provide correct parameters/options for the pipeline!\n";
374 $local_status = open(INPUT, $inpF);
375 if ($local_status == 0) { print "Fail to open file $inpF!\n"; exit(-1); }
379 while ($line = <INPUT>) {
381 my @local_arr = split(/\t/, $line);
382 push(@results, \@local_arr);
387 $local_status = open(OUTPUT, ">$outF");
388 if ($local_status == 0) { print "Fail to create file $outF!\n"; exit(-1); }
390 my $n = scalar(@results);
391 my $m = scalar(@{$results[0]});
396 for (my $i = 0; $i < $n; $i++) {
397 if ($_[0] eq "isoform") { push(@out_arr, $transcript_title[$i]); }
398 elsif ($_[0] eq "gene") { push(@out_arr, $gene_title[$i]); }
399 else { print "A bug on 'collectResults' is detected!\n"; exit(-1); }
401 print OUTPUT "@out_arr\n";
403 for (my $i = 0; $i < $m; $i++) {
405 for (my $j = 0; $j < $n; $j++) { push(@out_arr, $results[$j][$i]); }
406 print OUTPUT "@out_arr\n";
413 sub showVersionInfo {
414 open(INPUT, "$_[0]\WHAT_IS_NEW");
426 rsem-calculate-expression
432 rsem-calculate-expression [options] upstream_read_file(s) reference_name sample_name
433 rsem-calculate-expression [options] --paired-end upstream_read_file(s) downstream_read_file(s) reference_name sample_name
434 rsem-calculate-expression [options] --sam/--bam [--paired-end] input reference_name sample_name
442 =item B<upstream_read_files(s)>
444 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.
446 =item B<downstream_read_file(s)>
448 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.
452 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.
454 =item B<reference_name>
456 The name of the reference used. The user must have run 'rsem-prepare-reference' with this reference_name before running this program.
460 The name of the sample analyzed. All output files are prefixed by this name (e.g., sample_name.genes.results)
468 =item B<--paired-end>
470 Input reads are paired-end reads. (Default: off)
472 =item B<--no-qualities>
474 Input reads do not contain quality scores. (Default: off)
476 =item B<--strand-specific>
478 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)
482 Input file is in SAM format. (Default: off)
486 Input file is in BAM format. (Default: off)
488 =item B<--sam-header-info> <file>
490 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: "")
492 =item B<-p/--num-threads> <int>
494 Number of threads to use. Both Bowtie and expression estimation will use this many threads. (Default: 1)
496 =item B<--no-bam-output>
498 Do not output any BAM file. (Default: off)
500 =item B<--output-genome-bam>
502 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)
504 =item B<--sampling-for-bam>
506 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)
510 Calculate 95% credibility intervals and posterior mean estimates. (Default: off)
512 =item B<--seed-length> <int>
514 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)
516 =item B<--tag> <string>
518 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: "")
520 =item B<--bowtie-path> <path>
522 The path to the bowtie executables. (Default: the path to the bowtie executables is assumed to be in the user's PATH environment variable)
524 =item B<--bowtie-n> <int>
526 (Bowtie parameter) max # of mismatches in the seed. (Range: 0-3, Default: 2)
528 =item B<--bowtie-e> <int>
530 (Bowtie parameter) max sum of mismatch quality scores across the alignment. (Default: 99999999)
532 =item B<--bowtie-m> <int>
534 (Bowtie parameter) suppress all alignments for a read if > <int> valid alignments exist. (Default: 200)
536 =item B<--bowtie-chunkmbs> <int>
538 (Bowtie parameter) memory allocated for best first alignment calculation (Default: 0 - use bowtie's default)
540 =item B<--phred33-quals>
542 Input quality scores are encoded as Phred+33. (Default: on)
544 =item B<--phred64-quals>
546 Input quality scores are encoded as Phred+64 (default for GA Pipeline ver. >= 1.3). (Default: off)
548 =item B<--solexa-quals>
550 Input quality scores are solexa encoded (from GA Pipeline ver. < 1.3). (Default: off)
552 =item B<--forward-prob> <double>
554 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)
556 =item B<--fragment-length-min> <int>
558 Minimum read/insert length allowed. This is also the value for the bowtie -I option. (Default: 1)
560 =item B<--fragment-length-max> <int>
562 Maximum read/insert length allowed. This is also the value for the bowtie -X option. (Default: 1000)
564 =item B<--fragment-length-mean> <double>
566 (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)
568 =item B<--fragment-length-sd> <double>
570 (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>)
572 =item B<--estimate-rspd>
574 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)
576 =item B<--num-rspd-bins> <int>
578 Number of bins in the RSPD. Only relevant when '--estimate-rspd' is specified. Use of the default setting is recommended. (Default: 20)
580 =item B<--ci-memory> <int>
582 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)
584 =item B<--keep-intermediate-files>
586 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)
588 =item B<--temporary-folder> <string>
590 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)
594 Output time consumed by each step of RSEM to 'sample_name.time'. (Default: off)
598 Suppress the output of logging information. (Default: off)
602 Show help information.
608 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.
610 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:
612 convert-sam-for-rsem /ref/mouse_125 input.sam -o input_for_rsem.sam
614 For details, please refer to 'convert-sam-for-rsem's documentation page.
616 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 '*'.
618 The user must run 'rsem-prepare-reference' with the appropriate reference before using this program.
620 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.
622 Please note that some of the default values for the Bowtie parameters are not the same as those defined for Bowtie itself.
624 The temporary directory and all intermediate files will be removed when RSEM finishes unless '--keep-intermediate-files' is specified.
626 With the '--calc-ci' option, 95% credibility intervals and posterior mean estimates will be calculated in addition to maximum likelihood estimates.
632 =item B<sample_name.isoforms.results>
634 File containing isoform level expression estimates. The first line
635 contains column names separated by the tab character. The format of
636 each line in the rest of this file is:
638 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]
640 Fields are separated by the tab character. Fields within "[]" are only
641 presented if '--calc-ci' is set.
643 'transcript_id' is the transcript name of this transcript. 'gene_id'
644 is the gene name of the gene which this transcript belongs to (denote
645 this gene as its parent gene). If no gene information is provided,
646 'gene_id' and 'transcript_id' are the same.
648 'length' is this transcript's sequence length (poly(A) tail is not
649 counted). 'effective_length' counts only the positions that can
650 generate a valid fragment. If no poly(A) tail is added,
651 'effective_length' is equal to transcript length - mean fragment
652 length + 1. If one transcript's effective length is less than 1, this
653 transcript's both effective length and abundance estimates are set to
656 'expected_count' is the sum of the posterior probability of each read
657 comes from this transcript over all reads. Because 1) each read
658 aligning to this transcript has a probability of being generated from
659 background noise; 2) RSEM may filter some alignable low quality reads,
660 the sum of expected counts for all transcript are generally less than
661 the total number of reads aligned.
663 'TPM' stands for Transcripts Per Million. It is a relative measure of
664 transcript abundance. The sum of all transcripts' TPM is 1
665 million. 'FPKM' stands for Fragments Per Kilobase of transcript per
666 Million mapped reads. It is another relative measure of transcript
667 abundance. If we define l_bar be the mean transcript length in a
668 sample, which can be calculated as
670 l_bar = \sum_i TPM_i / 10^6 * effective_length_i (i goes through every transcript),
672 the following equation is hold:
674 FPKM_i = 10^3 / l_bar * TPM_i.
676 We can see that the sum of FPKM is not a constant across samples.
678 'IsoPct' stands for isoform percentage. It is the percentage of this
679 transcript's abandunce over its parent gene's abandunce. If its parent
680 gene has only one isoform or the gene information is not provided,
681 this field will be set to 100.
683 'pme_expected_count', 'pme_TPM', 'pme_FPKM' are posterior mean
684 estimates calculated by RSEM's Gibbs sampler. 'IsoPct_from_pme_TPM' is
685 the isoform percentage calculated from 'pme_TPM' values.
687 'TPM_ci_lower_bound', 'TPM_ci_upper_bound', 'FPKM_ci_lower_bound' and
688 'FPKM_ci_upper_bound' are lower(l) and upper(u) bounds of 95%
689 credibility intervals for TPM and FPKM values. The bounds are
690 inclusive (i.e. [l, u]).
692 =item B<sample_name.genes.results>
694 File containing gene level expression estimates. The first line
695 contains column names separated by the tab character. The format of
696 each line in the rest of this file is:
698 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]
700 Fields are separated by the tab character. Fields within "[]" are only
701 presented if '--calc-ci' is set.
703 'transcript_id(s)' is a comma-separated list of transcript_ids
704 belonging to this gene. If no gene information is provided, 'gene_id'
705 and 'transcript_id(s)' are identical (the 'transcript_id').
707 A gene's 'length' and 'effective_length' are
708 defined as the weighted average of its transcripts' lengths and
709 effective lengths (weighted by 'IsoPct'). A gene's abundance estimates
710 are just the sum of its transcripts' abundance estimates.
712 =item B<sample_name.transcript.bam, sample_name.transcript.sorted.bam and sample_name.transcript.sorted.bam.bai>
714 Only generated when --no-bam-output is not specified.
716 'sample_name.transcript.bam' is a BAM-formatted file of read
717 alignments in transcript coordinates. The MAPQ field of each alignment
718 is set to min(100, floor(-10 * log10(1.0 - w) + 0.5)), where w is the
719 posterior probability of that alignment being the true mapping of a
720 read. In addition, RSEM pads a new tag ZW:f:value, where value is a
721 single precision floating number representing the posterior
722 probability. Because this file contains all alignment lines produced
723 by bowtie or user-specified aligners, it can also be used as a
724 replacement of the aligner generated BAM/SAM file. For paired-end
725 reads, if one mate has alignments but the other does not, this file
726 marks the alignable mate as "unmappable" (flag bit 0x4) and appends an
727 optional field "Z0:A:!".
729 'sample_name.transcript.sorted.bam' and
730 'sample_name.transcript.sorted.bam.bai' are the sorted BAM file and
731 indices generated by samtools (included in RSEM package).
733 =item B<sample_name.genome.bam, sample_name.genome.sorted.bam and sample_name.genome.sorted.bam.bai>
735 Only generated when --no-bam-output is not specified and --output-genome-bam is specified.
737 'sample_name.genome.bam' is a BAM-formatted file of read alignments in
738 genomic coordinates. Alignments of reads that have identical genomic
739 coordinates (i.e., alignments to different isoforms that share the
740 same genomic region) are collapsed into one alignment. The MAPQ field
741 of each alignment is set to min(100, floor(-10 * log10(1.0 - w) +
742 0.5)), where w is the posterior probability of that alignment being
743 the true mapping of a read. In addition, RSEM pads a new tag
744 ZW:f:value, where value is a single precision floating number
745 representing the posterior probability. If an alignment is spliced, a
746 XS:A:value tag is also added, where value is either '+' or '-'
747 indicating the strand of the transcript it aligns to.
749 'sample_name.genome.sorted.bam' and 'sample_name.genome.sorted.bam.bai' are the
750 sorted BAM file and indices generated by samtools (included in RSEM package).
752 =item B<sample_name.time>
754 Only generated when --time is specified.
756 It contains time (in seconds) consumed by aligning reads, estimating expression levels and calculating credibility intervals.
758 =item B<sample_name.stat>
760 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.
766 Assume the path to the bowtie executables is in the user's PATH environment variable. Reference files are under '/ref' with name 'mouse_125'.
768 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:
770 rsem-calculate-expression --phred64-quals \
772 --output-genome-bam \
777 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:
779 rsem-calculate-expression -p 8 \
784 mmliver_paired_end_quals
786 3) '/data/mmliver.fa', single-end reads without quality scores. We want to use 8 threads:
788 rsem-calculate-expression -p 8 \
792 mmliver_single_without_quals
794 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:
796 rsem-calculate-expression --bowtie-path /sw/bowtie \
798 --fragment-length-mean 150.0 \
799 --fragment-length-sd 35.0 \
801 --output-genome-bam \
808 5) '/data/mmliver_paired_end_quals.bam', paired-end reads with quality scores. We want to use 8 threads:
810 rsem-calculate-expression --paired-end \
813 /data/mmliver_paired_end_quals.bam \
815 mmliver_paired_end_quals