13 my $CONFIDENCE = 0.95;
16 my $NMB = 1024; # default
20 my $read_type = 1; # default, single end with qual
47 my $genBamF = 1; # default is generating transcript bam file
48 my $genGenomeBamF = 0;
56 my $keep_intermediate_files = 0;
58 my $strand_specific = 0;
61 my ($time_start, $time_end, $time_alignment, $time_rsem, $time_ci) = (0, 0, 0, 0, 0);
63 GetOptions("keep-intermediate-files" => \$keep_intermediate_files,
64 "no-qualities" => \$no_qual,
65 "paired-end" => \$paired_end,
66 "strand-specific" => \$strand_specific,
69 "sam-header-info=s" => \$fn_list,
71 "seed-length=i" => \$L,
72 "bowtie-path=s" => \$bowtie_path,
75 "bowtie-m=i" => \$maxHits,
76 "phred33-quals" => \$phred33,
77 "phred64-quals" => \$phred64, #solexa1.3-quals" => \$phred64,
78 "solexa-quals" => \$solexa,
79 "forward-prob=f" => \$probF,
80 "fragment-length-min=i" => \$minL,
81 "fragment-length-max=i" => \$maxL,
82 "fragment-length-mean=f" => \$mean,
83 "fragment-length-sd=f" => \$sd,
84 "estimate-rspd" => \$estRSPD,
85 "num-rspd-bins=i" => \$B,
86 "p|num-threads=i" => \$nThreads,
87 "output-genome-bam" => \$genGenomeBamF,
88 "sampling-for-bam" => \$sampling,
89 "calc-ci" => \$calcCI,
90 "ci-memory=i" => \$NMB,
93 "h|help" => \$help) or pod2usage(-exitval => 2, -verbose => 2);
95 pod2usage(-verbose => 2) if ($help == 1);
98 #check parameters and options
100 if ($is_sam || $is_bam) {
101 pod2usage(-msg => "Invalid number of arguments!", -exitval => 2, -verbose => 2) if (scalar(@ARGV) != 3);
102 pod2usage(-msg => "--sam and --bam cannot be active at the same time!", -exitval => 2, -verbose => 2) if ($is_sam == 1&& $is_bam == 1);
103 pod2usage(-msg => "--bowtie-path, --bowtie-n, --bowtie-e, --bowtie-m, --phred33-quals, --phred64-quals or --solexa-quals cannot be set if input is SAM/BAM format!", -exitval => 2, -verbose => 2) if ($bowtie_path ne "" || $C != 2 || $E != 99999999 || $maxHits != 200 || $phred33 || $phred64 || $solexa);
106 pod2usage(-msg => "Invalid number of arguments!", -exitval => 2, -verbose => 2) if (!$paired_end && scalar(@ARGV) != 3 || $paired_end && scalar(@ARGV) != 4);
107 pod2usage(-msg => "Only one of --phred33-quals --phred64-quals/--solexa1.3-quals --solexa-suqls can be active!", -exitval => 2, -verbose => 2) if ($phred33 + $phred64 + $solexa > 1);
108 podwusage(-msg => "--sam , --bam or --sam-header-info cannot be set if use bowtie aligner to produce alignments!", -exitval => 2, -verbose => 2) if ($is_sam || $is_bam || $fn_list ne "");
111 pod2usage(-msg => "Forward probability should be in [0, 1]!", -exitval => 2, -verbose => 2) if ($probF < 0 || $probF > 1);
112 pod2usage(-msg => "Min fragment length should be at least 1!", -exitval => 2, -verbose => 2) if ($minL < 1);
113 pod2usage(-msg => "Min fragment length should be smaller or equal to max fragment length!", -exitval => 2, -verbose => 2) if ($minL > $maxL);
114 pod2usage(-msg => "The memory allocated for calculating credibility intervals should be at least 1 MB!\n", -exitval => 2, -verbose => 2) if ($NMB < 1);
115 pod2usage(-msg => "Number of threads should be at least 1!\n", -exitval => 2, -verbose => 2) if ($nThreads < 1);
116 pod2usage(-msg => "Seed length should be at least 25!\n", -exitval => 2, -verbose => 2) if ($L < 25);
117 pod2usage(-msg => "--sampling-for-bam cannot be specified if --out-bam is not specified!\n", -exitval => 2, -verbose => 2) if ($sampling && !$genBamF);
119 if ($strand_specific) { $probF = 1.0; }
125 my ($refName, $sampleName, $sampleToken, $temp_dir, $stat_dir, $imdName) = ();
129 if ($no_qual) { $read_type = 2; }
130 else { $read_type = 3; }
133 if ($no_qual) { $read_type = 0; }
134 else { $read_type = 1; }
137 if (scalar(@ARGV) == 3) {
138 if ($is_sam || $is_bam) { $inpF = $ARGV[0]; }
139 else {$mate1_list = $ARGV[0]; }
141 $sampleName = $ARGV[2];
144 $mate1_list = $ARGV[0];
145 $mate2_list = $ARGV[1];
147 $sampleName = $ARGV[3];
150 if ($genGenomeBamF) {
151 open(INPUT, "$refName.ti");
152 my $line = <INPUT>; chomp($line);
154 my ($M, $type) = split(/ /, $line);
155 pod2usage(-msg => "No genome information provided, so genome bam file cannot be generated!\n", -exitval => 2, -verbose => 2) if ($type != 0);
158 my $pos = rindex($sampleName, '/');
159 if ($pos < 0) { $sampleToken = $sampleName; }
160 else { $sampleToken = substr($sampleName, $pos + 1); }
162 $temp_dir = "$sampleName.temp";
163 $stat_dir = "$sampleName.stat";
165 if (!(-d $temp_dir) && !mkdir($temp_dir)) { print "Fail to create folder $temp_dir.\n"; exit(-1); }
166 if (!(-d $stat_dir) && !mkdir($stat_dir)) { print "Fail to create folder $stat_dir.\n"; exit(-1); }
168 $imdName = "$temp_dir/$sampleToken";
170 if (!$is_sam && !$is_bam && $phred33 + $phred64 + $solexa == 0) { $phred33 = 1; }
172 my ($mate_minL, $mate_maxL) = (1, $maxL);
174 if ($bowtie_path ne "") { $bowtie_path .= "/"; }
176 my ($fn, $dir, $suf) = fileparse($0);
179 if (!$is_sam && !$is_bam) {
180 $command = $bowtie_path."bowtie";
181 if ($read_type == 0 || $read_type == 2) { $command .= " -f"; }
182 else { $command .= " -q"; }
184 if ($phred33) { $command .= " --phred33-quals"; }
185 elsif ($phred64) { $command .= " --phred64-quals"; }
186 elsif ($solexa) { $command .= " --solexa-quals"; }
187 else { print "Oh, no!!!"; exit(2); }
189 $command .= " -n $C -e $E -l $L";
191 if ($read_type == 2 || $read_type == 3) { $command .= " -I $minL -X $maxL"; }
193 if ($strand_specific || $probF == 1.0) { $command .= " --norc"; }
194 elsif ($probF == 0.0) { $command .= " --nofw"; }
196 $command .= " -p $nThreads -a -m $maxHits -S";
197 if ($quiet) { $command .= " --quiet"; }
199 $command .= " $refName";
200 if ($read_type == 0 || $read_type == 1) {
201 $command .= " $mate1_list";
204 $command .= " -1 $mate1_list -2 $mate2_list";
207 $command .= " | gzip > $imdName.sam.gz";
209 if ($mTime) { $time_start = time(); }
211 &runCommand($command);
213 if ($mTime) { $time_end = time(); $time_alignment = $time_end - $time_start; }
215 $inpF = "$imdName.sam.gz";
216 $is_sam = 1; # output of bowtie is a sam file
219 if ($mTime) { $time_start = time(); }
221 $command = $dir."rsem-parse-alignments $refName $sampleName $sampleToken";
224 if ($is_sam) { $samInpType = "s"; }
225 elsif ($is_bam) { $samInpType = "b"; }
227 $command .= " $samInpType $inpF -t $read_type";
228 if ($fn_list ne "") { $command .= " -l $fn_list"; }
229 if ($tagName ne "") { $command .= " -tag $tagName"; }
230 if ($quiet) { $command .= " -q"; }
232 &runCommand($command);
234 $command = $dir."rsem-build-read-index $gap";
236 case 0 { $command .= " 0 $quiet $imdName\_alignable.fa"; }
237 case 1 { $command .= " 1 $quiet $imdName\_alignable.fq"; }
238 case 2 { $command .= " 0 $quiet $imdName\_alignable_1.fa $imdName\_alignable_2.fa"; }
239 case 3 { $command .= " 1 $quiet $imdName\_alignable_1.fq $imdName\_alignable_2.fq"; }
241 &runCommand($command);
243 my $doesOpen = open(OUTPUT, ">$imdName.mparams");
244 if ($doesOpen == 0) { print "Cannot generate $imdName.mparams!\n"; exit(-1); }
245 print OUTPUT "$minL $maxL\n";
246 print OUTPUT "$probF\n";
247 print OUTPUT "$estRSPD\n";
249 print OUTPUT "$mate_minL $mate_maxL\n";
250 print OUTPUT "$mean $sd\n";
254 $command = $dir."rsem-run-em $refName $read_type $sampleName $sampleToken -p $nThreads";
256 $command .= " -b $samInpType $inpF";
257 if ($fn_list ne "") { $command .= " 1 $fn_list"; }
258 else { $command .= " 0"; }
259 if ($sampling) { $command .= " --sampling"; }
261 if ($calcCI) { $command .= " --gibbs-out"; }
262 if ($quiet) { $command .= " -q"; }
264 &runCommand($command);
267 $command = $dir."sam/samtools sort $sampleName.transcript.bam $sampleName.transcript.sorted";
268 &runCommand($command);
269 $command = $dir."sam/samtools index $sampleName.transcript.sorted.bam";
270 &runCommand($command);
272 if ($genGenomeBamF) {
273 $command = $dir."rsem-tbam2gbam $refName $sampleName.transcript.bam $sampleName.genome.bam";
274 &runCommand($command);
275 $command = $dir."sam/samtools sort $sampleName.genome.bam $sampleName.genome.sorted";
276 &runCommand($command);
277 $command = $dir."sam/samtools index $sampleName.genome.sorted.bam";
278 &runCommand($command);
282 &collectResults("$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
283 &collectResults("$imdName.gene_res", "$sampleName.genes.results"); # gene level
285 if ($mTime) { $time_end = time(); $time_rsem = $time_end - $time_start; }
287 if ($mTime) { $time_start = time(); }
290 $command = $dir."rsem-run-gibbs $refName $sampleName $sampleToken $BURNIN $CHAINLEN $SAMPLEGAP";
291 # $command .= " -p $nThreads";
292 if ($quiet) { $command .= " -q"; }
293 &runCommand($command);
295 system("mv $sampleName.isoforms.results $imdName.isoforms.results.bak1");
296 system("mv $sampleName.genes.results $imdName.genes.results.bak1");
297 &collectResults("$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
298 &collectResults("$imdName.gene_res", "$sampleName.genes.results"); # gene level
300 $command = $dir."rsem-calculate-credibility-intervals $refName $sampleName $sampleToken $CONFIDENCE $NSPC $NMB";
301 if ($quiet) { $command .= " -q"; }
302 &runCommand($command);
304 system("mv $sampleName.isoforms.results $imdName.isoforms.results.bak2");
305 system("mv $sampleName.genes.results $imdName.genes.results.bak2");
306 &collectResults("$imdName.iso_res", "$sampleName.isoforms.results"); # isoform level
307 &collectResults("$imdName.gene_res", "$sampleName.genes.results"); # gene level
310 if ($mTime) { $time_end = time(); $time_ci = $time_end - $time_start; }
312 if ($mTime) { $time_start = time(); }
314 if (!$keep_intermediate_files) {
315 &runCommand("rm -rf $temp_dir", "Fail to delete the temporary folder!");
318 if ($mTime) { $time_end = time(); }
321 open(OUTPUT, ">$sampleName.time");
322 print OUTPUT "Alignment: $time_alignment s.\n";
323 print OUTPUT "RSEM: $time_rsem s.\n";
324 print OUTPUT "CI: $time_ci s.\n";
325 my $time_del = $time_end - $time_start;
326 print OUTPUT "Delete: $time_del s.\n";
333 my $status = system($_[0]);
336 if (scalar(@_) > 1) { $errmsg = $_[1]; }
337 else { $errmsg = "\"$command\" failed! Plase check if you provide correct parameters/options for the pipeline!"; }
348 my (@results, @ids) = ();
355 $local_status = open(INPUT, $inpF);
356 if ($local_status == 0) { print "Fail to open file $inpF!\n"; exit(-1); }
361 while ($line = <INPUT>) {
364 my @local_arr = split(/\t/, $line);
365 if ($cnt == 4) { @ids = @local_arr; }
366 else { push(@results, \@local_arr); }
369 push(@results, \@ids);
372 $local_status = open(OUTPUT, ">$outF");
373 if ($local_status == 0) { print "Fail to create file $outF!\n"; exit(-1); }
375 my $n = scalar(@results);
376 my $m = scalar(@{$results[0]});
377 for (my $i = 0; $i < $m; $i++) {
379 for (my $j = 0; $j < $n; $j++) { push(@out_arr, $results[$j][$i]); }
381 print OUTPUT "@out_arr\n";
391 rsem-calculate-expression
397 rsem-calculate-expression [options] upstream_read_file(s) reference_name sample_name
398 rsem-calculate-expression [options] --paired-end upstream_read_file(s) downstream_read_file(s) reference_name sample_name
399 rsem-calculate-expression [options] --sam/--bam [--paired-end] input reference_name sample_name
407 =item B<upstream_read_files(s)>
409 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.
411 =item B<downstream_read_file(s)>
413 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.
417 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.
419 =item B<reference_name>
421 The name of the reference used. The user must have run 'rsem-prepare-reference' with this reference_name before running this program.
425 The name of the sample analyzed. All output files are prefixed by this name (e.g., sample_name.genes.results)
433 =item B<--paired-end>
435 Input reads are paired-end reads. (Default: off)
437 =item B<--no-qualities>
439 Input reads do not contain quality scores. (Default: off)
441 =item B<--strand-specific>
443 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)
447 Input file is in SAM format. (Default: off)
451 Input file is in BAM format. (Default: off)
453 =item B<--sam-header-info> <file>
455 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: "")
457 =item B<-p/--num-threads> <int>
459 Number of threads to use. Both Bowtie and expression estimation will use this many threads. (Default: 1)
461 =item B<--output-genome-bam>
463 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)
465 =item B<--sampling-for-bam>
467 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)
471 Calculate 95% credibility intervals and posterior mean estimates. (Default: off)
473 =item B<--seed-length> <int>
475 Seed length used by the read aligner. Providing the correct value for this parameter is important for RSEM's accuracy if the data are single-end reads. If RSEM runs Bowtie, it uses this value for Bowtie's seed length parameter. The minimum value is 25. Any read with its or at least one of its mates' (for paired-end reads) length less than 25 will be ignored. (Default: 25)
477 =item B<--tag> <string>
479 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: "")
481 =item B<--bowtie-path> <path>
483 The path to the bowtie executables. (Default: the path to the bowtie executables is assumed to be in the user's PATH environment variable)
485 =item B<--bowtie-n> <int>
487 (Bowtie parameter) max # of mismatches in the seed. (Range: 0-3, Default: 2)
489 =item B<--bowtie-e> <int>
491 (Bowtie parameter) max sum of mismatch quality scores across the alignment. (Default: 99999999)
493 =item B<--bowtie-m> <int>
495 (Bowtie parameter) suppress all alignments for a read if > <int> valid alignments exist. (Default: 200)
497 =item B<--phred33-quals>
499 Input quality scores are encoded as Phred+33. (Default: on)
501 =item B<--phred64-quals>
503 Input quality scores are encoded as Phred+64 (default for GA Pipeline ver. >= 1.3). (Default: off)
505 =item B<--solexa-quals>
507 Input quality scores are solexa encoded (from GA Pipeline ver. < 1.3). (Default: off)
509 =item B<--forward-prob> <double>
511 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)
513 =item B<--fragment-length-min> <int>
515 Minimum read/insert length allowed. This is also the value for the bowtie -I option. (Default: 1)
517 =item B<--fragment-length-max> <int>
519 Maximum read/insert length allowed. This is also the value for the bowtie -X option. (Default: 1000)
521 =item B<--fragment-length-mean> <double>
523 (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)
525 =item B<--fragment-length-sd> <double>
527 (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>)
529 =item B<--estimate-rspd>
531 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)
533 =item B<--num-rspd-bins> <int>
535 Number of bins in the RSPD. Only relevant when '--estimate-rspd' is specified. Use of the default setting is recommended. (Default: 20)
537 =item B<--ci-memory> <int>
539 Amount of memory (in MB) RSEM is allowed to use for computing credibility intervals. (Default: 1024)
541 =item B<--keep-intermediate-files>
543 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)
547 Suppress the output of logging information. (Default: off)
551 Show help information.
557 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.
559 One simple way to make the alignment file (e.g. input.sam) satisfying RSEM's requirements (assuming the aligner used put mates in a paired-end read adjacent) is to use the following command:
561 sort -k 1,1 -s input.sam > input.sorted.sam
563 The SAM/BAM format RSEM uses is v1.4. However, it is compatible with old SAM/BAM format.
565 The user must run 'rsem-prepare-reference' with the appropriate reference before using this program.
567 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.
569 Please note that some of the default values for the Bowtie parameters are not the same as those defined for Bowtie itself.
571 The temporary directory and all intermediate files will be removed when RSEM finishes unless '--keep-intermediate-files' is specified.
573 With the "--calc-ci" option, 95% credibility intervals and posterior mean estimates will be calculated in addition to maximum likelihood estimates.
579 =item B<sample_name.genes.results>
581 File containing gene level expression estimates. The format of each
582 line in this file is:
584 gene_id expected_counts tau_value [pmc_value tau_pme_value tau_ci_lower_bound tau_ci_upper_bound] transcript_id_list
586 Fields are separated by the tab character. Fields within "[]" are only
587 presented if '--calc-ci' is set. pme stands for posterior mean
588 estimation. pmc stands for posterior mean counts. ci_lower_bound(l)
589 means the lower bound of the credibility intervals, ci_upper_bound(u)
590 means the upper bound of the credibility intervals. So the credibility
591 interval is [l, u]. 'transcript_id_list' is a space-separated list of
592 transcript_ids belonging to the gene. If no gene information is
593 provided, this file has the same content as
594 'sample_name.isoforms.results'.
596 =item B<sample_name.isoforms.results>
598 File containing isoform level expression values. The format of each
599 line in this file is:
601 transcript_id expected_counts tau_value [pmc_value tau_pme_value tau_ci_lower_bound tau_ci_upper_bound] gene_id
603 Fields are separated by the tab character. 'gene_id' is the gene_id of
604 the gene which this transcript belongs to. If no gene information is
605 provided, 'gene_id' and 'transcript_id' are the same.
607 =item B<sample_name.transcript.bam, sample_name.transcript.sorted.bam and sample_name.transcript.sorted.bam.bai>
609 'sample_name.transcript.bam' is a BAM-formatted file of read
610 alignments in transcript coordinates. The MAPQ field of each alignment
611 is set to min(100, floor(-10 * log10(1.0 - w) + 0.5)), where w is the
612 posterior probability of that alignment being the true mapping of a
613 read. In addition, RSEM pads a new tag ZW:f:value, where value is a
614 single precision floating number representing the posterior
617 'sample_name.transcript.sorted.bam' and
618 'sample_name.transcript.sorted.bam.bai' are the sorted BAM file and
619 indices generated by samtools (included in RSEM package).
621 =item B<sample_name.genome.bam, sample_name.genome.sorted.bam and sample_name.genome.sorted.bam.bai>
623 Only generated when --output-genome-bam is specified.
625 'sample_name.genome.bam' is a BAM-formatted file of read alignments in
626 genomic coordinates. Alignments of reads that have identical genomic
627 coordinates (i.e., alignments to different isoforms that share the
628 same genomic region) are collapsed into one alignment. The MAPQ field
629 of each alignment is set to min(100, floor(-10 * log10(1.0 - w) +
630 0.5)), where w is the posterior probability of that alignment being
631 the true mapping of a read. In addition, RSEM pads a new tag
632 ZW:f:value, where value is a single precision floating number
633 representing the posterior probability. If an alignment is spliced, a
634 XS:A:value tag is also added, where value is either '+' or '-'
635 indicating the strand of the transcript it aligns to.
637 'sample_name.genome.sorted.bam' and 'sample_name.genome.sorted.bam.bai' are the
638 sorted BAM file and indices generated by samtools (included in RSEM package).
640 =item B<sample_name.stat>
642 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.
648 Assume the path to the bowtie executables is in the user's PATH environment variable. Reference files are under '/ref' with name 'mm9'.
650 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:
652 rsem-calculate-expression --phred64-quals \
654 --output-genome-bam \
659 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:
661 rsem-calculate-expression -p 8 \
666 mmliver_paired_end_quals
668 3) '/data/mmliver.fa', single-end reads without quality scores. We want to use 8 threads:
670 rsem-calculate-expression -p 8 \
674 mmliver_single_without_quals
676 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:
678 rsem-calculate-expression --bowtie-path /sw/bowtie \
680 --fragment-length-mean 150.0 \
681 --fragment-length-sd 35.0 \
683 --output-genome-bam \
690 5) '/data/mmliver_paired_end_quals.bam', paired-end reads with quality scores. We want to use 8 threads:
692 rsem-calculate-expression --paired-end \
695 /data/mmliver_paired_end_quals.bam \
697 mmliver_paired_end_quals