## <a name="introduction"></a> Introduction
RSEM is a software package for estimating gene and isoform expression
-levels from RNA-Seq data. The new RSEM package (rsem-1.x) provides an
-user-friendly interface, supports threads for parallel computation of
-the EM algorithm, single-end and paired-end read data, quality scores,
-variable-length reads and RSPD estimation. It can also generate
-genomic-coordinate BAM files and UCSC wiggle files for
-visualization. In addition, it provides posterior mean and 95%
-credibility interval estimates for expression levels. For
-visualization, it can also generate transcript-coordinate BAM files
-and visualize them and also models learned.
+levels from RNA-Seq data. The RSEM package provides an user-friendly
+interface, supports threads for parallel computation of the EM
+algorithm, single-end and paired-end read data, quality scores,
+variable-length reads and RSPD estimation. In addition, it provides
+posterior mean and 95% credibility interval estimates for expression
+levels. For visualization, It can generate BAM and Wiggle files in
+both transcript-coordinate and genomic-coordinate. Genomic-coordinate
+files can be visualized by both UCSC Genome browser and Broad
+Institute's Integrative Genomics Viewer (IGV). Transcript-coordinate
+files can be visualized by IGV. RSEM also has its own scripts to
+generate transcript read depth plots in pdf format. The unique feature
+of RSEM is, the read depth plots can be stacked, with read depth
+contributed to unique reads shown in black and contributed to
+multi-reads shown in red. In addition, models learned from data can
+also be visualized. Last but not least, RSEM contains a simulator.
## <a name="compilation"></a> Compilation & Installation
By default, RSEM automates the alignment of reads to reference
transcripts using the Bowtie alignment program. To use an alternative
alignment program, align the input reads against the file
-'reference_name.idx.fa' generated by 'rsem-prepare-reference', and format
-the alignment output in SAM or BAM format. Then, instead of providing
-reads to 'rsem-calculate-expression', specify the '--sam' or '--bam' option
-and provide the SAM or BAM file as an argument. When using an
-alternative aligner, you may also want to provide the '--no-bowtie' option
-to 'rsem-prepare-reference' so that the Bowtie indices are not built.
-
-Some aligners' (other than Bowtie) output might need to be converted
-so that RSEM can use. For conversion, please run
+'reference_name.idx.fa' generated by 'rsem-prepare-reference', and
+format the alignment output in SAM or BAM format. Then, instead of
+providing reads to 'rsem-calculate-expression', specify the '--sam' or
+'--bam' option and provide the SAM or BAM file as an argument. When
+using an alternative aligner, you may also want to provide the
+'--no-bowtie' option to 'rsem-prepare-reference' so that the Bowtie
+indices are not built.
+
+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. To check if your SAM/BAM file satisfy the requirements,
+please run
+
+ rsem-sam-validator <input.sam/input.bam>
+
+If your file does not satisfy the requirements, you can use
+'convert-sam-for-rsem' to convert it into a BAM file which RSEM can
+process. Please run
- convert-sam-for-rsem --help
+ convert-sam-for-rsem --help
to get usage information or visit the [convert-sam-for-rsem
documentation
For UCSC genome browser, please refer to the [UCSC custom track help page](http://genome.ucsc.edu/goldenPath/help/customTrack.html).
-For integrative genomics viewer, please refer to the [IGV home page](http://www.broadinstitute.org/software/igv/home).
+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.
#### c) Generating Transcript Wiggle Plots
RSEM uses the [Boost C++](http://www.boost.org) and
[samtools](http://samtools.sourceforge.net) libraries.
+We thank earonesty for contributing patches.
+
## <a name="license"></a> License
RSEM is licensed under the [GNU General Public License v3](http://www.gnu.org/licenses/gpl-3.0.html).