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 * [Acknowledgements](#acknowledgements)
21 ## Introduction <a name="introduction"></a>
23 RSEM is a software package for estimating gene and isoform expression
24 levels from RNA-Seq data. The new RSEM package (rsem-1.x) provides an
25 user-friendly interface, supports threads for parallel computation of
26 the EM algorithm, single-end and paired-end read data, quality scores,
27 variable-length reads and RSPD estimation. It can also generate
28 genomic-coordinate BAM files and UCSC wiggle files for visualization. In
29 addition, it provides posterior mean and 95% credibility interval
30 estimates for expression levels.
32 ## Compilation & Installation <a name="compilation"></a>
34 To compile RSEM, simply run
38 To install, simply put the rsem directory in your environment's PATH
43 To take advantage of RSEM's built-in support for the Bowtie alignment
44 program, you must have [Bowtie](http://bowtie-bio.sourceforge.net) installed.
46 ## <a name="usage"></a> Usage
49 ### I. Preparing Reference Sequences
51 RSEM can extract reference transcripts from a genome if you provide it
52 with gene annotations in a GTF file. Alternatively, you can provide
53 RSEM with transcript sequences directly.
55 Please note that GTF files generated from the UCSC Table Browser do not
56 contain isoform-gene relationship information. However, if you use the
57 UCSC Genes annotation track, this information can be recovered by
58 downloading the knownIsoforms.txt file for the appropriate genome.
60 To prepare the reference sequences, you should run the
61 'rsem-prepare-reference' program. Run
63 rsem-prepare-reference --help
65 to get usage information or visit the [rsem-prepare-reference
66 documentation page](http://deweylab.biostat.wisc.edu/rsem/rsem-prepare-reference.html).
68 ### II. Calculating Expression Values
70 To calculate expression values, you should run the
71 'rsem-calculate-expression' program. Run
73 rsem-calculate-expression --help
75 to get usage information or visit the [rsem-calculate-expression
76 documentation page](http://deweylab.biostat.wisc.edu/rsem/rsem-calculate-expression.html).
78 #### Calculating expression values from single-end data
80 For single-end models, users have the option of providing a fragment
81 length distribution via the --fragment-length-mean and
82 --fragment-length-sd options. The specification of an accurate fragment
83 length distribution is important for the accuracy of expression level
84 estimates from single-end data. If the fragment length mean and sd are
85 not provided, RSEM will not take a fragment length distribution into
88 #### Using an alternative aligner
90 By default, RSEM automates the alignment of reads to reference
91 transcripts using the Bowtie alignment program. To use an alternative
92 alignment program, align the input reads against the file
93 'reference_name.idx.fa' generated by rsem-prepare-reference, and format
94 the alignment output in SAM or BAM format. Then, instead of providing
95 reads to rsem-calculate-expression, specify the --sam or --bam option
96 and provide the SAM or BAM file as an argument. When using an
97 alternative aligner, you may also want to provide the --no-bowtie option
98 to rsem-prepare-reference so that the Bowtie indices are not built.
100 ### III. Visualization
102 RSEM contains a version of samtools in the 'sam' subdirectory. When
103 users specify the --out-bam option RSEM will produce three files:
104 'sample_name.bam', the unsorted BAM file, 'sample_name.sorted.bam' and
105 'sample_name.sorted.bam.bai' the sorted BAM file and indices generated
106 by the samtools included.
108 #### a) Generating a UCSC Wiggle file
110 A wiggle plot representing the expected number of reads overlapping
111 each position in the genome can be generated from the sorted BAM file
112 output. To generate the wiggle plot, run the 'rsem-bam2wig' program on
113 the 'sample_name.sorted.bam' file.
117 rsem-bam2wig bam_input wig_output wiggle_name
119 bam_input: sorted bam file
120 wig_output: output file name, e.g. output.wig
121 wiggle_name: the name the user wants to use for this wiggle plot
123 #### b) Loading a BAM and/or Wiggle file into the UCSC Genome Browser
125 Refer to the [UCSC custom track help page](http://genome.ucsc.edu/goldenPath/help/customTrack.html).
127 ## Example <a name="example"></a>
129 Suppose we download the mouse genome from UCSC Genome Browser. We will
130 use a reference_name of 'mm9'. We have a FASTQ-formatted file,
131 'mmliver.fq', containing single-end reads from one sample, which we call
132 'mmliver_single_quals'. We want to estimate expression values by using
133 the single-end model with a fragment length distribution. We know that
134 the fragment length distribution is approximated by a normal
135 distribution with a mean of 150 and a standard deviation of 35. We wish
136 to generate 95% credibility intervals in addition to maximum likelihood
137 estimates. RSEM will be allowed 1G of memory for the credibility
138 interval calculation. We will visualize the probabilistic read mappings
141 The commands for this scenario are as follows:
143 rsem-prepare-reference --gtf mm9.gtf --mapping knownIsoforms.txt --bowtie-path /sw/bowtie /data/mm9 /ref/mm9
144 rsem-calculate-expression --bowtie-path /sw/bowtie --phred64-quals --fragment-length-mean 150.0 --fragment-length-sd 35.0 -p 8 --out-bam --calc-ci --memory-allocate 1024 /data/mmliver.fq /ref/mm9 mmliver_single_quals
145 rsem-bam2wig mmliver_single_quals.sorted.bam mmliver_single_quals.sorted.wig mmliver_single_quals
147 ## Simulation <a name="simulation"></a>
151 rsem-simulate-reads reference_name estimated_model_file estimated_isoform_results theta0 N output_name [-q]
153 estimated_model_file: File containing model parameters. Generated by
154 rsem-calculate-expression.
155 estimated_isoform_results: File containing isoform expression levels.
156 Generated by rsem-calculate-expression.
157 theta0: fraction of reads that are "noise" (not derived from a transcript).
158 N: number of reads to simulate.
159 output_name: prefix for all output files.
160 [-q] : set it will stop outputting intermediate information.
164 output_name.fa if single-end without quality score;
165 output_name.fq if single-end with quality score;
166 output_name_1.fa & output_name_2.fa if paired-end without quality
168 output_name_1.fq & output_name_2.fq if paired-end with quality score.
170 output_name.sim.isoforms.results, output_name.sim.genes.results : Results estimated based on sample values.
172 ## Acknowledgements <a name="acknowledgements"></a>
174 RSEM uses randomc.h and mersenne.cpp from
175 <http://lxnt.info/rng/randomc.htm> for random number generation. RSEM
176 also uses the [Boost C++](http://www.boost.org) and
177 [samtools](http://samtools.sourceforge.net) libraries.
179 ## License <a name="license"></a>
181 RSEM is licensed under the [GNU General Public License v3](http://www.gnu.org/licenses/gpl-3.0.html).