13 my $CONFIDENCE = 0.95;
16 my $NMB = 1024; # default
20 my $read_type = 1; # default, single end with qual
27 my $chunkMbs = 0; # 0 = use bowtie default
48 my $genBamF = 1; # default is generating transcript bam file
49 my $genGenomeBamF = 0;
52 my $var_opt = 0; # temporarily, only for internal use
58 my $keep_intermediate_files = 0;
60 my $strand_specific = 0;
63 my ($time_start, $time_end, $time_alignment, $time_rsem, $time_ci) = (0, 0, 0, 0, 0);
65 GetOptions("keep-intermediate-files" => \$keep_intermediate_files,
66 "no-qualities" => \$no_qual,
67 "paired-end" => \$paired_end,
68 "strand-specific" => \$strand_specific,
71 "sam-header-info=s" => \$fn_list,
73 "seed-length=i" => \$L,
74 "bowtie-path=s" => \$bowtie_path,
77 "bowtie-m=i" => \$maxHits,
78 "bowtie-chunkmbs=i" => \$chunkMbs,
79 "phred33-quals" => \$phred33,
80 "phred64-quals" => \$phred64, #solexa1.3-quals" => \$phred64,
81 "solexa-quals" => \$solexa,
82 "forward-prob=f" => \$probF,
83 "fragment-length-min=i" => \$minL,
84 "fragment-length-max=i" => \$maxL,
85 "fragment-length-mean=f" => \$mean,
86 "fragment-length-sd=f" => \$sd,
87 "estimate-rspd" => \$estRSPD,
88 "num-rspd-bins=i" => \$B,
89 "p|num-threads=i" => \$nThreads,
90 "no-bam-output" => sub { $genBamF = 0; },
91 "output-genome-bam" => \$genGenomeBamF,
92 "sampling-for-bam" => \$sampling,
94 "calc-ci" => \$calcCI,
95 "ci-memory=i" => \$NMB,
98 "h|help" => \$help) or pod2usage(-exitval => 2, -verbose => 2);
100 pod2usage(-verbose => 2) if ($help == 1);
103 #check parameters and options
105 if ($is_sam || $is_bam) {
106 pod2usage(-msg => "Invalid number of arguments!", -exitval => 2, -verbose => 2) if (scalar(@ARGV) != 3);
107 pod2usage(-msg => "--sam and --bam cannot be active at the same time!", -exitval => 2, -verbose => 2) if ($is_sam == 1&& $is_bam == 1);
108 pod2usage(-msg => "--bowtie-path, --bowtie-n, --bowtie-e, --bowtie-m, --phred33-quals, --phred64-quals or --solexa-quals cannot be set if input is SAM/BAM format!", -exitval => 2, -verbose => 2) if ($bowtie_path ne "" || $C != 2 || $E != 99999999 || $maxHits != 200 || $phred33 || $phred64 || $solexa);
111 pod2usage(-msg => "Invalid number of arguments!", -exitval => 2, -verbose => 2) if (!$paired_end && scalar(@ARGV) != 3 || $paired_end && scalar(@ARGV) != 4);
112 pod2usage(-msg => "Only one of --phred33-quals --phred64-quals/--solexa1.3-quals --solexa-suqls can be active!", -exitval => 2, -verbose => 2) if ($phred33 + $phred64 + $solexa > 1);
113 podwusage(-msg => "--sam , --bam or --sam-header-info cannot be set if use bowtie aligner to produce alignments!", -exitval => 2, -verbose => 2) if ($is_sam || $is_bam || $fn_list ne "");
116 pod2usage(-msg => "Forward probability should be in [0, 1]!", -exitval => 2, -verbose => 2) if ($probF < 0 || $probF > 1);
117 pod2usage(-msg => "Min fragment length should be at least 1!", -exitval => 2, -verbose => 2) if ($minL < 1);
118 pod2usage(-msg => "Min fragment length should be smaller or equal to max fragment length!", -exitval => 2, -verbose => 2) if ($minL > $maxL);
119 pod2usage(-msg => "The memory allocated for calculating credibility intervals should be at least 1 MB!\n", -exitval => 2, -verbose => 2) if ($NMB < 1);
120 pod2usage(-msg => "Number of threads should be at least 1!\n", -exitval => 2, -verbose => 2) if ($nThreads < 1);
121 pod2usage(-msg => "Seed length should be at least 5!\n", -exitval => 2, -verbose => 2) if ($L < 5);
122 pod2usage(-msg => "--sampling-for-bam cannot be specified if --no-bam-output is specified!\n", -exitval => 2, -verbose => 2) if ($sampling && !$genBamF);
123 pod2usage(-msg => "--output-genome-bam cannot be specified if --no-bam-output is specified!\n", -exitval => 2, -verbose => 2) if ($genGenomeBamF && !$genBamF);
125 if ($L < 25) { print "Warning: the seed length set is less than 25! This is only allowed if the references are not added poly(A) tails.\n"; }
127 if ($strand_specific) { $probF = 1.0; }
133 my ($refName, $sampleName, $sampleToken, $temp_dir, $stat_dir, $imdName) = ();
137 if ($no_qual) { $read_type = 2; }
138 else { $read_type = 3; }
141 if ($no_qual) { $read_type = 0; }
142 else { $read_type = 1; }
145 if (scalar(@ARGV) == 3) {
146 if ($is_sam || $is_bam) { $inpF = $ARGV[0]; }
147 else {$mate1_list = $ARGV[0]; }
149 $sampleName = $ARGV[2];
152 $mate1_list = $ARGV[0];
153 $mate2_list = $ARGV[1];
155 $sampleName = $ARGV[3];
158 if ($genGenomeBamF) {
159 open(INPUT, "$refName.ti");
160 my $line = <INPUT>; chomp($line);
162 my ($M, $type) = split(/ /, $line);
163 pod2usage(-msg => "No genome information provided, so genome bam file cannot be generated!\n", -exitval => 2, -verbose => 2) if ($type != 0);
166 my $pos = rindex($sampleName, '/');
167 if ($pos < 0) { $sampleToken = $sampleName; }
168 else { $sampleToken = substr($sampleName, $pos + 1); }
170 $temp_dir = "$sampleName.temp";
171 $stat_dir = "$sampleName.stat";
173 if (!(-d $temp_dir) && !mkdir($temp_dir)) { print "Fail to create folder $temp_dir.\n"; exit(-1); }
174 if (!(-d $stat_dir) && !mkdir($stat_dir)) { print "Fail to create folder $stat_dir.\n"; exit(-1); }
176 $imdName = "$temp_dir/$sampleToken";
178 if (!$is_sam && !$is_bam && !$no_qual && ($phred33 + $phred64 + $solexa == 0)) { $phred33 = 1; }
180 my ($mate_minL, $mate_maxL) = (1, $maxL);
182 if ($bowtie_path ne "") { $bowtie_path .= "/"; }
184 my ($fn, $dir, $suf) = fileparse($0);
187 if (!$is_sam && !$is_bam) {
188 $command = $bowtie_path."bowtie";
189 if ($no_qual) { $command .= " -f"; }
190 else { $command .= " -q"; }
192 if ($phred33) { $command .= " --phred33-quals"; }
193 elsif ($phred64) { $command .= " --phred64-quals"; }
194 elsif ($solexa) { $command .= " --solexa-quals"; }
196 $command .= " -n $C -e $E -l $L";
197 if ($read_type == 2 || $read_type == 3) { $command .= " -I $minL -X $maxL"; }
198 if ($chunkMbs > 0) { $command .= " --chunkmbs $chunkMbs"; }
200 if ($strand_specific || $probF == 1.0) { $command .= " --norc"; }
201 elsif ($probF == 0.0) { $command .= " --nofw"; }
203 $command .= " -p $nThreads -a -m $maxHits -S";
204 if ($quiet) { $command .= " --quiet"; }
206 $command .= " $refName";
207 if ($read_type == 0 || $read_type == 1) {
208 $command .= " $mate1_list";
211 $command .= " -1 $mate1_list -2 $mate2_list";
214 $command .= " | gzip > $sampleName.sam.gz";
216 if ($mTime) { $time_start = time(); }
218 &runCommand($command);
220 if ($mTime) { $time_end = time(); $time_alignment = $time_end - $time_start; }
222 $inpF = "$sampleName.sam.gz";
223 $is_sam = 1; # output of bowtie is a sam file
226 if ($mTime) { $time_start = time(); }
228 $command = $dir."rsem-parse-alignments $refName $sampleName $sampleToken";
231 if ($is_sam) { $samInpType = "s"; }
232 elsif ($is_bam) { $samInpType = "b"; }
234 $command .= " $samInpType $inpF -t $read_type";
235 if ($fn_list ne "") { $command .= " -l $fn_list"; }
236 if ($tagName ne "") { $command .= " -tag $tagName"; }
237 if ($quiet) { $command .= " -q"; }
239 &runCommand($command);
241 $command = $dir."rsem-build-read-index $gap";
243 case 0 { $command .= " 0 $quiet $imdName\_alignable.fa"; }
244 case 1 { $command .= " 1 $quiet $imdName\_alignable.fq"; }
245 case 2 { $command .= " 0 $quiet $imdName\_alignable_1.fa $imdName\_alignable_2.fa"; }
246 case 3 { $command .= " 1 $quiet $imdName\_alignable_1.fq $imdName\_alignable_2.fq"; }
248 &runCommand($command);
250 my $doesOpen = open(OUTPUT, ">$imdName.mparams");
251 if ($doesOpen == 0) { print "Cannot generate $imdName.mparams!\n"; exit(-1); }
252 print OUTPUT "$minL $maxL\n";
253 print OUTPUT "$probF\n";
254 print OUTPUT "$estRSPD\n";
256 print OUTPUT "$mate_minL $mate_maxL\n";
257 print OUTPUT "$mean $sd\n";
261 $command = $dir."rsem-run-em $refName $read_type $sampleName $sampleToken -p $nThreads";
263 $command .= " -b $samInpType $inpF";
264 if ($fn_list ne "") { $command .= " 1 $fn_list"; }
265 else { $command .= " 0"; }
266 if ($sampling) { $command .= " --sampling"; }
268 if ($calcCI) { $command .= " --gibbs-out"; }
269 if ($quiet) { $command .= " -q"; }
271 &runCommand($command);
274 $command = $dir."sam/samtools sort $sampleName.transcript.bam $sampleName.transcript.sorted";
275 &runCommand($command);
276 $command = $dir."sam/samtools index $sampleName.transcript.sorted.bam";
277 &runCommand($command);
279 if ($genGenomeBamF) {
280 $command = $dir."rsem-tbam2gbam $refName $sampleName.transcript.bam $sampleName.genome.bam";
281 &runCommand($command);
282 $command = $dir."sam/samtools sort $sampleName.genome.bam $sampleName.genome.sorted";
283 &runCommand($command);
284 $command = $dir."sam/samtools index $sampleName.genome.sorted.bam";
285 &runCommand($command);
289 &collectResults("$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
290 &collectResults("$imdName.gene_res", "$sampleName.genes.results"); # gene level
292 if ($mTime) { $time_end = time(); $time_rsem = $time_end - $time_start; }
294 if ($mTime) { $time_start = time(); }
296 if ($calcCI || $var_opt) {
297 $command = $dir."rsem-run-gibbs $refName $sampleName $sampleToken $BURNIN $NCV $SAMPLEGAP";
298 $command .= " -p $nThreads";
299 if ($var_opt) { $command .= " --var"; }
300 if ($quiet) { $command .= " -q"; }
301 &runCommand($command);
305 system("mv $sampleName.isoforms.results $imdName.isoforms.results.bak1");
306 system("mv $sampleName.genes.results $imdName.genes.results.bak1");
307 &collectResults("$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
308 &collectResults("$imdName.gene_res", "$sampleName.genes.results"); # gene level
310 $command = $dir."rsem-calculate-credibility-intervals $refName $sampleName $sampleToken $CONFIDENCE $NCV $NSPC $NMB";
311 $command .= " -p $nThreads";
312 if ($quiet) { $command .= " -q"; }
313 &runCommand($command);
315 system("mv $sampleName.isoforms.results $imdName.isoforms.results.bak2");
316 system("mv $sampleName.genes.results $imdName.genes.results.bak2");
317 &collectResults("$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
318 &collectResults("$imdName.gene_res", "$sampleName.genes.results"); # gene level
321 if ($mTime) { $time_end = time(); $time_ci = $time_end - $time_start; }
323 if ($mTime) { $time_start = time(); }
325 if (!$keep_intermediate_files) {
326 &runCommand("rm -rf $temp_dir", "Fail to delete the temporary folder!");
329 if ($mTime) { $time_end = time(); }
332 open(OUTPUT, ">$sampleName.time");
333 print OUTPUT "Aligning reads: $time_alignment s.\n";
334 print OUTPUT "Estimating expression levels: $time_rsem s.\n";
335 print OUTPUT "Calculating credibility intervals: $time_ci s.\n";
336 my $time_del = $time_end - $time_start;
337 # print OUTPUT "Delete: $time_del s.\n";
344 my $status = system($_[0]);
347 if (scalar(@_) > 1) { $errmsg = $_[1]; }
348 else { $errmsg = "\"$command\" failed! Plase check if you provide correct parameters/options for the pipeline!"; }
359 my (@results, @ids) = ();
366 $local_status = open(INPUT, $inpF);
367 if ($local_status == 0) { print "Fail to open file $inpF!\n"; exit(-1); }
372 while ($line = <INPUT>) {
375 my @local_arr = split(/\t/, $line);
376 if ($cnt == 4) { @ids = @local_arr; }
377 else { push(@results, \@local_arr); }
380 push(@results, \@ids);
383 $local_status = open(OUTPUT, ">$outF");
384 if ($local_status == 0) { print "Fail to create file $outF!\n"; exit(-1); }
386 my $n = scalar(@results);
387 my $m = scalar(@{$results[0]});
388 for (my $i = 0; $i < $m; $i++) {
390 for (my $j = 0; $j < $n; $j++) { push(@out_arr, $results[$j][$i]); }
392 print OUTPUT "@out_arr\n";
402 rsem-calculate-expression
408 rsem-calculate-expression [options] upstream_read_file(s) reference_name sample_name
409 rsem-calculate-expression [options] --paired-end upstream_read_file(s) downstream_read_file(s) reference_name sample_name
410 rsem-calculate-expression [options] --sam/--bam [--paired-end] input reference_name sample_name
418 =item B<upstream_read_files(s)>
420 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.
422 =item B<downstream_read_file(s)>
424 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.
428 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.
430 =item B<reference_name>
432 The name of the reference used. The user must have run 'rsem-prepare-reference' with this reference_name before running this program.
436 The name of the sample analyzed. All output files are prefixed by this name (e.g., sample_name.genes.results)
444 =item B<--paired-end>
446 Input reads are paired-end reads. (Default: off)
448 =item B<--no-qualities>
450 Input reads do not contain quality scores. (Default: off)
452 =item B<--strand-specific>
454 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)
458 Input file is in SAM format. (Default: off)
462 Input file is in BAM format. (Default: off)
464 =item B<--sam-header-info> <file>
466 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: "")
468 =item B<-p/--num-threads> <int>
470 Number of threads to use. Both Bowtie and expression estimation will use this many threads. (Default: 1)
472 =item B<--no-bam-output>
474 Do not output any BAM file. (Default: off)
476 =item B<--output-genome-bam>
478 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)
480 =item B<--sampling-for-bam>
482 When RSEM generates a BAM file, instead of outputing all alignments a read has with their posterior probabilities, one alignment is sampled and outputed according to the posterior probabilities. If the sampling result is that the read comes from the "noise" transcript, nothing is outputed. (Default: off)
486 Calculate 95% credibility intervals and posterior mean estimates. (Default: off)
488 =item B<--seed-length> <int>
490 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)
492 =item B<--tag> <string>
494 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: "")
496 =item B<--bowtie-path> <path>
498 The path to the bowtie executables. (Default: the path to the bowtie executables is assumed to be in the user's PATH environment variable)
500 =item B<--bowtie-n> <int>
502 (Bowtie parameter) max # of mismatches in the seed. (Range: 0-3, Default: 2)
504 =item B<--bowtie-e> <int>
506 (Bowtie parameter) max sum of mismatch quality scores across the alignment. (Default: 99999999)
508 =item B<--bowtie-m> <int>
510 (Bowtie parameter) suppress all alignments for a read if > <int> valid alignments exist. (Default: 200)
512 =item B<--bowtie-chunkmbs> <int>
514 (Bowtie parameter) memory allocated for best first alignment calculation (Default: 0 - use bowtie's default)
516 =item B<--phred33-quals>
518 Input quality scores are encoded as Phred+33. (Default: on)
520 =item B<--phred64-quals>
522 Input quality scores are encoded as Phred+64 (default for GA Pipeline ver. >= 1.3). (Default: off)
524 =item B<--solexa-quals>
526 Input quality scores are solexa encoded (from GA Pipeline ver. < 1.3). (Default: off)
528 =item B<--forward-prob> <double>
530 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)
532 =item B<--fragment-length-min> <int>
534 Minimum read/insert length allowed. This is also the value for the bowtie -I option. (Default: 1)
536 =item B<--fragment-length-max> <int>
538 Maximum read/insert length allowed. This is also the value for the bowtie -X option. (Default: 1000)
540 =item B<--fragment-length-mean> <double>
542 (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)
544 =item B<--fragment-length-sd> <double>
546 (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>)
548 =item B<--estimate-rspd>
550 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)
552 =item B<--num-rspd-bins> <int>
554 Number of bins in the RSPD. Only relevant when '--estimate-rspd' is specified. Use of the default setting is recommended. (Default: 20)
556 =item B<--ci-memory> <int>
558 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)
560 =item B<--keep-intermediate-files>
562 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)
566 Output time consumed by each step of RSEM to 'sample_name.time'. (Default: off)
570 Suppress the output of logging information. (Default: off)
574 Show help information.
580 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.
582 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:
584 convert-sam-for-rsem /ref/mouse_125 input.sam -o input_for_rsem.sam
586 For details, please refer to 'convert-sam-for-rsem's documentation page.
588 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 '*'.
590 The user must run 'rsem-prepare-reference' with the appropriate reference before using this program.
592 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.
594 Please note that some of the default values for the Bowtie parameters are not the same as those defined for Bowtie itself.
596 The temporary directory and all intermediate files will be removed when RSEM finishes unless '--keep-intermediate-files' is specified.
598 With the '--calc-ci' option, 95% credibility intervals and posterior mean estimates will be calculated in addition to maximum likelihood estimates.
604 =item B<sample_name.genes.results>
606 File containing gene level expression estimates. The format of each
607 line in this file is:
609 gene_id expected_counts tau_value [pmc_value tau_pme_value tau_ci_lower_bound tau_ci_upper_bound] transcript_id_list
611 Fields are separated by the tab character. Fields within "[]" are only
612 presented if '--calc-ci' is set. pme stands for posterior mean
613 estimation. pmc stands for posterior mean counts. ci_lower_bound(l)
614 means the lower bound of the credibility intervals, ci_upper_bound(u)
615 means the upper bound of the credibility intervals. So the credibility
616 interval is [l, u]. 'transcript_id_list' is a space-separated list of
617 transcript_ids belonging to the gene. If no gene information is
618 provided, this file has the same content as
619 'sample_name.isoforms.results'.
621 =item B<sample_name.isoforms.results>
623 File containing isoform level expression values. The format of each
624 line in this file is:
626 transcript_id expected_counts tau_value [pmc_value tau_pme_value tau_ci_lower_bound tau_ci_upper_bound] gene_id
628 Fields are separated by the tab character. 'gene_id' is the gene_id of
629 the gene which this transcript belongs to. If no gene information is
630 provided, 'gene_id' and 'transcript_id' are the same.
632 =item B<sample_name.transcript.bam, sample_name.transcript.sorted.bam and sample_name.transcript.sorted.bam.bai>
634 Only generated when --no-bam-output is not specified.
636 'sample_name.transcript.bam' is a BAM-formatted file of read
637 alignments in transcript coordinates. The MAPQ field of each alignment
638 is set to min(100, floor(-10 * log10(1.0 - w) + 0.5)), where w is the
639 posterior probability of that alignment being the true mapping of a
640 read. In addition, RSEM pads a new tag ZW:f:value, where value is a
641 single precision floating number representing the posterior
644 'sample_name.transcript.sorted.bam' and
645 'sample_name.transcript.sorted.bam.bai' are the sorted BAM file and
646 indices generated by samtools (included in RSEM package).
648 =item B<sample_name.genome.bam, sample_name.genome.sorted.bam and sample_name.genome.sorted.bam.bai>
650 Only generated when --no-bam-output is not specified and --output-genome-bam is specified.
652 'sample_name.genome.bam' is a BAM-formatted file of read alignments in
653 genomic coordinates. Alignments of reads that have identical genomic
654 coordinates (i.e., alignments to different isoforms that share the
655 same genomic region) are collapsed into one alignment. The MAPQ field
656 of each alignment is set to min(100, floor(-10 * log10(1.0 - w) +
657 0.5)), where w is the posterior probability of that alignment being
658 the true mapping of a read. In addition, RSEM pads a new tag
659 ZW:f:value, where value is a single precision floating number
660 representing the posterior probability. If an alignment is spliced, a
661 XS:A:value tag is also added, where value is either '+' or '-'
662 indicating the strand of the transcript it aligns to.
664 'sample_name.genome.sorted.bam' and 'sample_name.genome.sorted.bam.bai' are the
665 sorted BAM file and indices generated by samtools (included in RSEM package).
667 =item B<sample_name.sam.gz>
669 Only generated when the input files are raw reads instead of SAM/BAM format files
671 It is the gzipped SAM output produced by bowtie aligner.
673 =item B<sample_name.time>
675 Only generated when --time is specified.
677 It contains time (in seconds) consumed by aligning reads, estimating expression levels and calculating credibility intervals.
679 =item B<sample_name.stat>
681 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.
687 Assume the path to the bowtie executables is in the user's PATH environment variable. Reference files are under '/ref' with name 'mouse_125'.
689 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:
691 rsem-calculate-expression --phred64-quals \
693 --output-genome-bam \
698 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:
700 rsem-calculate-expression -p 8 \
705 mmliver_paired_end_quals
707 3) '/data/mmliver.fa', single-end reads without quality scores. We want to use 8 threads:
709 rsem-calculate-expression -p 8 \
713 mmliver_single_without_quals
715 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:
717 rsem-calculate-expression --bowtie-path /sw/bowtie \
719 --fragment-length-mean 150.0 \
720 --fragment-length-sd 35.0 \
722 --output-genome-bam \
729 5) '/data/mmliver_paired_end_quals.bam', paired-end reads with quality scores. We want to use 8 threads:
731 rsem-calculate-expression --paired-end \
734 /data/mmliver_paired_end_quals.bam \
736 mmliver_paired_end_quals