4 [Bo Li](http://pages.cs.wisc.edu/~bli) \(bli at cs dot wisc dot edu\)
11 * [Introduction](#introduction)
12 * [Compilation & Installation](#compilation)
15 * [Simulation](#simulation)
16 * [Generate Transcript-to-Gene-Map from Trinity Output](#gen_trinity)
17 * [Differential Expression Analysis](#de)
19 * [Acknowledgements](#acknowledgements)
24 ## <a name="introduction"></a> Introduction
26 RSEM is a software package for estimating gene and isoform expression
27 levels from RNA-Seq data. The RSEM package provides an user-friendly
28 interface, supports threads for parallel computation of the EM
29 algorithm, single-end and paired-end read data, quality scores,
30 variable-length reads and RSPD estimation. In addition, it provides
31 posterior mean and 95% credibility interval estimates for expression
32 levels. For visualization, It can generate BAM and Wiggle files in
33 both transcript-coordinate and genomic-coordinate. Genomic-coordinate
34 files can be visualized by both UCSC Genome browser and Broad
35 Institute's Integrative Genomics Viewer (IGV). Transcript-coordinate
36 files can be visualized by IGV. RSEM also has its own scripts to
37 generate transcript read depth plots in pdf format. The unique feature
38 of RSEM is, the read depth plots can be stacked, with read depth
39 contributed to unique reads shown in black and contributed to
40 multi-reads shown in red. In addition, models learned from data can
41 also be visualized. Last but not least, RSEM contains a simulator.
43 ## <a name="compilation"></a> Compilation & Installation
45 To compile RSEM, simply run
49 To install, simply put the rsem directory in your environment's PATH
54 C++, Perl and R are required to be installed.
56 To take advantage of RSEM's built-in support for the Bowtie alignment
57 program, you must have [Bowtie](http://bowtie-bio.sourceforge.net) installed.
59 ## <a name="usage"></a> Usage
61 ### I. Preparing Reference Sequences
63 RSEM can extract reference transcripts from a genome if you provide it
64 with gene annotations in a GTF file. Alternatively, you can provide
65 RSEM with transcript sequences directly.
67 Please note that GTF files generated from the UCSC Table Browser do not
68 contain isoform-gene relationship information. However, if you use the
69 UCSC Genes annotation track, this information can be recovered by
70 downloading the knownIsoforms.txt file for the appropriate genome.
72 To prepare the reference sequences, you should run the
73 'rsem-prepare-reference' program. Run
75 rsem-prepare-reference --help
77 to get usage information or visit the [rsem-prepare-reference
78 documentation page](http://deweylab.biostat.wisc.edu/rsem/rsem-prepare-reference.html).
80 ### II. Calculating Expression Values
82 To calculate expression values, you should run the
83 'rsem-calculate-expression' program. Run
85 rsem-calculate-expression --help
87 to get usage information or visit the [rsem-calculate-expression
88 documentation page](http://deweylab.biostat.wisc.edu/rsem/rsem-calculate-expression.html).
90 #### Calculating expression values from single-end data
92 For single-end models, users have the option of providing a fragment
93 length distribution via the '--fragment-length-mean' and
94 '--fragment-length-sd' options. The specification of an accurate fragment
95 length distribution is important for the accuracy of expression level
96 estimates from single-end data. If the fragment length mean and sd are
97 not provided, RSEM will not take a fragment length distribution into
100 #### Using an alternative aligner
102 By default, RSEM automates the alignment of reads to reference
103 transcripts using the Bowtie alignment program. To use an alternative
104 alignment program, align the input reads against the file
105 'reference_name.idx.fa' generated by 'rsem-prepare-reference', and
106 format the alignment output in SAM or BAM format. Then, instead of
107 providing reads to 'rsem-calculate-expression', specify the '--sam' or
108 '--bam' option and provide the SAM or BAM file as an argument. When
109 using an alternative aligner, you may also want to provide the
110 '--no-bowtie' option to 'rsem-prepare-reference' so that the Bowtie
111 indices are not built.
113 RSEM requires all alignments of the same read group together. For
114 paired-end reads, RSEM also requires the two mates of any alignment be
115 adjacent. To check if your SAM/BAM file satisfy the requirements,
118 rsem-sam-validator <input.sam/input.bam>
120 If your file does not satisfy the requirements, you can use
121 'convert-sam-for-rsem' to convert it into a BAM file which RSEM can
124 convert-sam-for-rsem --help
126 to get usage information or visit the [convert-sam-for-rsem
128 page](http://deweylab.biostat.wisc.edu/rsem/convert-sam-for-rsem.html).
130 However, please note that RSEM does ** not ** support gapped
131 alignments. So make sure that your aligner does not produce alignments
132 with intersions/deletions. Also, please make sure that you use
133 'reference_name.idx.fa' , which is generated by RSEM, to build your
136 ### III. Visualization
138 RSEM contains a version of samtools in the 'sam' subdirectory. RSEM
139 will always produce three files:'sample_name.transcript.bam', the
140 unsorted BAM file, 'sample_name.transcript.sorted.bam' and
141 'sample_name.transcript.sorted.bam.bai' the sorted BAM file and
142 indices generated by the samtools included. All three files are in
143 transcript coordinates. When users specify the --output-genome-bam
144 option RSEM will produce three files: 'sample_name.genome.bam', the
145 unsorted BAM file, 'sample_name.genome.sorted.bam' and
146 'sample_name.genome.sorted.bam.bai' the sorted BAM file and indices
147 generated by the samtools included. All these files are in genomic
150 #### a) Converting transcript BAM file into genome BAM file
152 Normally, RSEM will do this for you via '--output-genome-bam' option
153 of 'rsem-calculate-expression'. However, if you have run
154 'rsem-prepare-reference' and use 'reference_name.idx.fa' to build
155 indices for your aligner, you can use 'rsem-tbam2gbam' to convert your
156 transcript coordinate BAM alignments file into a genomic coordinate
157 BAM alignments file without the need to run the whole RSEM
158 pipeline. Please note that 'rsem-prepare-reference' will convert all
159 'N' into 'G' by default for 'reference_name.idx.fa'. If you do not
160 want this to happen, please use '--no-ntog' option.
164 rsem-tbam2gbam reference_name unsorted_transcript_bam_input genome_bam_output
166 reference_name : The name of reference built by 'rsem-prepare-reference'
167 unsorted_transcript_bam_input : This file should satisfy: 1) the alignments of a same read are grouped together, 2) for any paired-end alignment, the two mates should be adjacent to each other, 3) this file should not be sorted by samtools
168 genome_bam_output : The output genomic coordinate BAM file's name
170 #### b) Generating a Wiggle file
172 A wiggle plot representing the expected number of reads overlapping
173 each position in the genome/transcript set can be generated from the
174 sorted genome/transcript BAM file output. To generate the wiggle
175 plot, run the 'rsem-bam2wig' program on the
176 'sample_name.genome.sorted.bam'/'sample_name.transcript.sorted.bam' file.
180 rsem-bam2wig sorted_bam_input wig_output wiggle_name [--no-fractional-weight]
182 sorted_bam_input : Input BAM format file, must be sorted
183 wig_output : Output wiggle file's name, e.g. output.wig
184 wiggle_name : The name of this wiggle plot
185 --no-fractional-weight : If this is set, RSEM will not look for "ZW" tag and each alignment appeared in the BAM file has weight 1. Set this if your BAM file is not generated by RSEM. Please note that this option must be at the end of the command line
187 #### c) Loading a BAM and/or Wiggle file into the UCSC Genome Browser or Integrative Genomics Viewer(IGV)
189 For UCSC genome browser, please refer to the [UCSC custom track help page](http://genome.ucsc.edu/goldenPath/help/customTrack.html).
191 For integrative genomics viewer, please refer to the [IGV home page](http://www.broadinstitute.org/software/igv/home). Note: Although IGV can generate read depth plot from the BAM file given, it cannot recognize "ZW" tag RSEM puts. Therefore IGV counts each alignment as weight 1 instead of the expected weight for the plot it generates. So we recommend to use the wiggle file generated by RSEM for read depth visualization.
193 Here are some guidance for visualizing transcript coordinate files using IGV:
195 1) Import the transcript sequences as a genome
197 Select File -> Import Genome, then fill in ID, Name and Fasta file. Fasta file should be 'reference_name.transcripts.fa'. After that, click Save button. Suppose ID is filled as 'reference_name', a file called 'reference_name.genome' will be generated. Next time, we can use: File -> Load Genome, then select 'reference_name.genome'.
199 2) Load visualization files
201 Select File -> Load from File, then choose one transcript coordinate visualization file generated by RSEM. IGV might require you to convert wiggle file to tdf file. You should use igvtools to perform this task. One way to perform the conversion is to use the following command:
203 igvtools tile reference_name.transcript.wig reference_name.transcript.tdf reference_name.genome
205 #### d) Generating Transcript Wiggle Plots
207 To generate transcript wiggle plots, you should run the
208 'rsem-plot-transcript-wiggles' program. Run
210 rsem-plot-transcript-wiggles --help
212 to get usage information or visit the [rsem-plot-transcript-wiggles
213 documentation page](http://deweylab.biostat.wisc.edu/rsem/rsem-plot-transcript-wiggles.html).
215 #### e) Visualize the model learned by RSEM
217 RSEM provides an R script, 'rsem-plot-model', for visulazing the model learned.
221 rsem-plot-model sample_name output_plot_file
223 sample_name: the name of the sample analyzed
224 output_plot_file: the file name for plots generated from the model. It is a pdf file
226 The plots generated depends on read type and user configuration. It
227 may include fragment length distribution, mate length distribution,
228 read start position distribution (RSPD), quality score vs observed
229 quality given a reference base, position vs percentage of sequencing
230 error given a reference base and histogram of reads with different
231 number of alignments.
233 fragment length distribution and mate length distribution: x-axis is fragment/mate length, y axis is the probability of generating a fragment/mate with the associated length
235 RSPD: Read Start Position Distribution. x-axis is bin number, y-axis is the probability of each bin. RSPD can be used as an indicator of 3' bias
237 Quality score vs. observed quality given a reference base: x-axis is Phred quality scores associated with data, y-axis is the "observed quality", Phred quality scores learned by RSEM from the data. Q = -10log_10(P), where Q is Phred quality score and P is the probability of sequencing error for a particular base
239 Position vs. percentage sequencing error given a reference base: x-axis is position and y-axis is percentage sequencing error
241 Histogram of reads with different number of alignments: x-axis is the number of alignments a read has and y-axis is the number of such reads. The inf in x-axis means number of reads filtered due to too many alignments
243 ## <a name="example"></a> Example
245 Suppose we download the mouse genome from UCSC Genome Browser. We
246 will use a reference_name of 'mouse_125'. We have a FASTQ-formatted file,
247 'mmliver.fq', containing single-end reads from one sample, which we
248 call 'mmliver_single_quals'. We want to estimate expression values by
249 using the single-end model with a fragment length distribution. We
250 know that the fragment length distribution is approximated by a normal
251 distribution with a mean of 150 and a standard deviation of 35. We
252 wish to generate 95% credibility intervals in addition to maximum
253 likelihood estimates. RSEM will be allowed 1G of memory for the
254 credibility interval calculation. We will visualize the probabilistic
255 read mappings generated by RSEM on UCSC genome browser. We will
256 generate a list of genes' transcript wiggle plots in 'output.pdf'. The
257 list is 'gene_ids.txt'. We will visualize the models learned in
258 'mmliver_single_quals.models.pdf'
260 The commands for this scenario are as follows:
262 rsem-prepare-reference --gtf mm9.gtf --mapping knownIsoforms.txt --bowtie-path /sw/bowtie /data/mm9 /ref/mouse_125
263 rsem-calculate-expression --bowtie-path /sw/bowtie --phred64-quals --fragment-length-mean 150.0 --fragment-length-sd 35.0 -p 8 --output-genome-bam --calc-ci --memory-allocate 1024 /data/mmliver.fq /ref/mouse_125 mmliver_single_quals
264 rsem-bam2wig mmliver_single_quals.sorted.bam mmliver_single_quals.sorted.wig mmliver_single_quals
265 rsem-plot-transcript-wiggles --gene-list --show-unique mmliver_single_quals gene_ids.txt output.pdf
266 rsem-plot-model mmliver_single_quals mmliver_single_quals.models.pdf
268 ## <a name="simulation"></a> Simulation
270 RSEM provides users the 'rsem-simulate-reads' program to simulate RNA-Seq data based on parameters learned from real data sets. Run
274 to get usage information or read the following subsections.
278 rsem-simulate-reads reference_name estimated_model_file estimated_isoform_results theta0 N output_name [-q]
280 __reference_name:__ The name of RSEM references, which should be already generated by 'rsem-prepare-reference'
282 __estimated_model_file:__ This file describes how the RNA-Seq reads will be sequenced given the expression levels. It determines what kind of reads will be simulated (single-end/paired-end, w/o quality score) and includes parameters for fragment length distribution, read start position distribution, sequencing error models, etc. Normally, this file should be learned from real data using 'rsem-calculate-expression'. The file can be found under the 'sample_name.stat' folder with the name of 'sample_name.model'
284 __estimated_isoform_results:__ This file contains expression levels for all isoforms recorded in the reference. It can be learned using 'rsem-calculate-expression' from real data. The corresponding file users want to use is 'sample_name.isoforms.results'. If simulating from user-designed expression profile is desired, start from a learned 'sample_name.isoforms.results' file and only modify the 'TPM' column. The simulator only reads the TPM column. But keeping the file format the same is required.
286 __theta0:__ This parameter determines the fraction of reads that are coming from background "noise" (instead of from a transcript). It can also be estimated using 'rsem-calculate-expression' from real data. Users can find it as the first value of the third line of the file 'sample_name.stat/sample_name.theta'.
288 __N:__ The total number of reads to be simulated. If 'rsem-calculate-expression' is executed on a real data set, the total number of reads can be found as the 4th number of the first line of the file 'sample_name.stat/sample_name.cnt'.
290 __output_name:__ Prefix for all output files.
292 __-q:__ Set it will stop outputting intermediate information.
296 output_name.sim.isoforms.results, output_name.sim.genes.results: Expression levels estimated by counting where each simulated read comes from.
298 output_name.fa if single-end without quality score;
299 output_name.fq if single-end with quality score;
300 output_name_1.fa & output_name_2.fa if paired-end without quality
302 output_name_1.fq & output_name_2.fq if paired-end with quality score.
304 **Format of the header line**: Each simulated read's header line encodes where it comes from. The header line has the format:
306 {>/@}_rid_dir_sid_pos[_insertL]
308 __{>/@}:__ Either '>' or '@' must appear. '>' appears if FASTA files are generated and '@' appears if FASTQ files are generated
310 __rid:__ Simulated read's index, numbered from 0
312 __dir:__ The direction of the simulated read. 0 refers to forward strand ('+') and 1 refers to reverse strand ('-')
314 __sid:__ Represent which transcript this read is simulated from. It ranges between 0 and M, where M is the total number of transcripts. If sid=0, the read is simulated from the background noise. Otherwise, the read is simulated from a transcript with index sid. Transcript sid's transcript name can be found in the 'transcript_id' column of the 'sample_name.isoforms.results' file (at line sid + 1, line 1 is for column names)
316 __pos:__ The start position of the simulated read in strand dir of transcript sid. It is numbered from 0
318 __insertL:__ Only appear for paired-end reads. It gives the insert length of the simulated read.
322 Suppose we want to simulate 50 millon single-end reads with quality scores and use the parameters learned from [Example](#example). In addition, we set theta0 as 0.2 and output_name as 'simulated_reads'. The command is:
324 rsem-simulate-reads /ref/mouse_125 mmliver_single_quals.stat/mmliver_single_quals.model mmliver_single_quals.isoforms.results 0.2 50000000 simulated_reads
326 ## <a name="gen_trinity"></a> Generate Transcript-to-Gene-Map from Trinity Output
328 For Trinity users, RSEM provides a perl script to generate transcript-to-gene-map file from the fasta file produced by Trinity.
332 extract-transcript-to-gene-map-from-trinity trinity_fasta_file map_file
334 trinity_fasta_file: the fasta file produced by trinity, which contains all transcripts assembled.
335 map_file: transcript-to-gene-map file's name.
337 ## <a name="de"></a> Differential Expression Analysis
339 Popular differential expression (DE) analysis tools such as edgeR and
340 DESeq do not take variance due to read mapping uncertainty into
341 consideration. Because read mapping ambiguity is prevalent among
342 isoforms and de novo assembled transcripts, these tools are not ideal
343 for DE detection in such conditions.
345 EBSeq, an empirical Bayesian DE analysis tool developed in UW-Madison,
346 can take variance due to read mapping ambiguity into consideration by
347 grouping isoforms with parent gene's number of isoforms. In addition,
348 it is more robust to outliers. For more information about EBSeq
349 (including the paper describing their method), please visit [EBSeq's
350 website](http://www.biostat.wisc.edu/~ningleng/EBSeq_Package).
353 RSEM includes EBSeq in its folder named 'EBSeq'. To use it, first type
357 to compile the EBSeq related codes.
359 EBSeq requires gene-isoform relationship for its isoform DE
360 detection. However, for de novo assembled transcriptome, it is hard to
361 obtain an accurate gene-isoform relationship. Instead, RSEM provides a
362 script 'rsem-generate-ngvector', which clusters transcripts based on
363 measures directly relating to read mappaing ambiguity. First, it
364 calcualtes the 'unmappability' of each transcript. The 'unmappability'
365 of a transcript is the ratio between the number of k mers with at
366 least one perfect match to other transcripts and the total number of k
367 mers of this transcript, where k is a parameter. Then, Ng vector is
368 generated by applying Kmeans algorithm to the 'unmappability' values
369 with number of clusters set as 3. This program will make sure the mean
370 'unmappability' scores for clusters are in ascending order. All
371 transcripts whose lengths are less than k are assigned to cluster
374 rsem-generate-ngvector --help
376 to get usage information or visit the [rsem-generate-ngvector
378 page](http://deweylab.biostat.wisc.edu/rsem/rsem-generate-ngvector.html).
380 If your reference is a de novo assembled transcript set, you should
381 run 'rsem-generate-ngvector' first. Then load the resulting
382 'output_name.ngvec' into R. For example, you can use
384 NgVec <- scan(file="output_name.ngvec", what=0, sep="\n")
386 . After that, set "NgVector = NgVec" for your differential expression
387 test (either 'EBTest' or 'EBMultiTest').
390 For users' convenience, RSEM also provides a script
391 'rsem-generate-data-matrix' to extract input matrix from expression
394 rsem-generate-data-matrix sampleA.[genes/isoforms].results sampleB.[genes/isoforms].results ... > output_name.counts.matrix
396 The results files are required to be either all gene level results or
397 all isoform level results. You can load the matrix into R by
399 IsoMat <- data.matrix(read.table(file="output_name.counts.matrix"))
401 before running either 'EBTest' or 'EBMultiTest'.
403 Lastly, RSEM provides two scripts, 'rsem-run-ebseq' and
404 'rsem-control-fdr', to help users find differential expressed
405 genes/transcripts. First, 'rsem-run-ebseq' calls EBSeq to calculate related statistics
406 for all genes/transcripts. Run
408 rsem-run-ebseq --help
410 to get usage information or visit the [rsem-run-ebseq documentation
411 page](http://deweylab.biostat.wisc.edu/rsem/rsem-run-ebseq.html). Second,
412 'rsem-control-fdr' takes 'rsem-run-ebseq' 's result and reports called
413 differentially expressed genes/transcripts by controlling the false
416 rsem-control-fdr --help
418 to get usage information or visit the [rsem-control-fdr documentation
419 page](http://deweylab.biostat.wisc.edu/rsem/rsem-control-fdr.html). These
420 two scripts can perform DE analysis on either 2 conditions or multiple
423 Please note that 'rsem-run-ebseq' and 'rsem-control-fdr' use EBSeq's
424 default parameters. For advanced use of EBSeq or information about how
425 EBSeq works, please refer to [EBSeq's
426 manual](http://www.bioconductor.org/packages/devel/bioc/vignettes/EBSeq/inst/doc/EBSeq_Vignette.pdf).
428 Questions related to EBSeq should
429 be sent to <a href="mailto:nleng@wisc.edu">Ning Leng</a>.
431 ## <a name="authors"></a> Authors
433 RSEM is developed by Bo Li, with substaintial technical input from Colin Dewey.
435 ## <a name="acknowledgements"></a> Acknowledgements
437 RSEM uses the [Boost C++](http://www.boost.org) and
438 [samtools](http://samtools.sourceforge.net) libraries. RSEM includes
439 [EBSeq](http://www.biostat.wisc.edu/~ningleng/EBSeq_Package/) for
440 differential expression analysis.
442 We thank earonesty for contributing patches.
444 We thank Han Lin for suggesting possible fixes.
446 ## <a name="license"></a> License
448 RSEM is licensed under the [GNU General Public License
449 v3](http://www.gnu.org/licenses/gpl-3.0.html).