make
+For cygwin users, please uncomment the 3rd and 7th line in
+'sam/Makefile' before you run 'make'.
+
+To compile EBSeq, which is included in the RSEM package, run
+
+ make ebseq
+
To install, simply put the rsem directory in your environment's PATH
variable.
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
+vpaired-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
generated by the samtools included. All these files are in genomic
coordinates.
-#### a) Generating a Wiggle file
+#### a) Converting transcript BAM file into genome BAM file
+
+Normally, RSEM will do this for you via '--output-genome-bam' option
+of 'rsem-calculate-expression'. However, if you have run
+'rsem-prepare-reference' and use 'reference_name.idx.fa' to build
+indices for your aligner, you can use 'rsem-tbam2gbam' to convert your
+transcript coordinate BAM alignments file into a genomic coordinate
+BAM alignments file without the need to run the whole RSEM
+pipeline. Please note that 'rsem-prepare-reference' will convert all
+'N' into 'G' by default for 'reference_name.idx.fa'. If you do not
+want this to happen, please use '--no-ntog' option.
+
+Usage:
+
+ rsem-tbam2gbam reference_name unsorted_transcript_bam_input genome_bam_output
+
+reference_name : The name of reference built by 'rsem-prepare-reference'
+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
+genome_bam_output : The output genomic coordinate BAM file's name
+
+#### b) Generating a Wiggle file
A wiggle plot representing the expected number of reads overlapping
each position in the genome/transcript set can be generated from the
sorted_bam_input : Input BAM format file, must be sorted
wig_output : Output wiggle file's name, e.g. output.wig
wiggle_name : the name of this wiggle plot
---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.
+--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
-#### b) Loading a BAM and/or Wiggle file into the UCSC Genome Browser or Integrative Genomics Viewer(IGV)
+#### c) Loading a BAM and/or Wiggle file into the UCSC Genome Browser or Integrative Genomics Viewer(IGV)
For UCSC genome browser, please refer to the [UCSC custom track help page](http://genome.ucsc.edu/goldenPath/help/customTrack.html).
1) Import the transcript sequences as a genome
-Select File -> Import Genome, then fill in ID, Name and Fasta file. Fasta file should be 'reference_name.idx.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'.
+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'.
2) Load visualization files
igvtools tile reference_name.transcript.wig reference_name.transcript.tdf reference_name.genome
-#### c) Generating Transcript Wiggle Plots
+#### d) Generating Transcript Wiggle Plots
To generate transcript wiggle plots, you should run the
'rsem-plot-transcript-wiggles' program. Run
to get usage information or visit the [rsem-plot-transcript-wiggles
documentation page](http://deweylab.biostat.wisc.edu/rsem/rsem-plot-transcript-wiggles.html).
-#### d) Visualize the model learned by RSEM
+#### e) Visualize the model learned by RSEM
RSEM provides an R script, 'rsem-plot-model', for visulazing the model learned.
DESeq do not take variance due to read mapping uncertainty into
consideration. Because read mapping ambiguity is prevalent among
isoforms and de novo assembled transcripts, these tools are not ideal
-for DE detection in such conditions.
+for DE detection in such conditions.
-**EBSeq**, an empirical Bayesian DE analysis tool developed in
-UW-Madison, can take variance due to read mapping ambiguity into
-consideration by grouping isoforms with parent gene's number of
-isoforms. In addition, it is more robust to outliers. For more
-information about EBSeq (including the paper describing their method),
-please visit <a
-href="http://www.biostat.wisc.edu/~ningleng/EBSeq_Package">EBSeq
-website</a>.
+EBSeq, an empirical Bayesian DE analysis tool developed in UW-Madison,
+can take variance due to read mapping ambiguity into consideration by
+grouping isoforms with parent gene's number of isoforms. In addition,
+it is more robust to outliers. For more information about EBSeq
+(including the paper describing their method), please visit [EBSeq's
+website](http://www.biostat.wisc.edu/~ningleng/EBSeq_Package).
-RSEM includes the newest version of EBSeq in its folder
-named 'EBSeq'. To use it, first type
+
+RSEM includes EBSeq in its folder named 'EBSeq'. To use it, first type
make ebseq
NgVec <- scan(file="output_name.ngvec", what=0, sep="\n")
-. After that, replace 'IsoNgTrun' with 'NgVec' in the second line of
-section 3.2.5 (Page 10) of EBSeq's vignette:
+. After that, set "NgVector = NgVec" for your differential expression
+test (either 'EBTest' or 'EBMultiTest').
- IsoEBres=EBTest(Data=IsoMat, NgVector=NgVec, ...)
For users' convenience, RSEM also provides a script
'rsem-generate-data-matrix' to extract input matrix from expression
IsoMat <- data.matrix(read.table(file="output_name.counts.matrix"))
-before running function 'EBTest'.
-
-At last, RSEM provides a R script, 'rsem-find-DE', which run EBSeq for
-you.
+before running either 'EBTest' or 'EBMultiTest'.
-Usage:
+Lastly, RSEM provides two scripts, 'rsem-run-ebseq' and
+'rsem-control-fdr', to help users find differential expressed
+genes/transcripts. First, 'rsem-run-ebseq' calls EBSeq to calculate related statistics
+for all genes/transcripts. Run
- rsem-find-DE data_matrix_file [--ngvector ngvector_file] number_sample_condition1 FDR_rate output_file
+ rsem-run-ebseq --help
-This script calls EBSeq to find differentially expressed genes/transcripts in two conditions.
+to get usage information or visit the [rsem-run-ebseq documentation
+page](http://deweylab.biostat.wisc.edu/rsem/rsem-run-ebseq.html). Second,
+'rsem-control-fdr' takes 'rsem-run-ebseq' 's result and reports called
+differentially expressed genes/transcripts by controlling the false
+discovery rate. Run
-data_matrix_file: m by n matrix containing expected counts, m is the number of transcripts/genes, n is the number of total samples.
-[--ngvector ngvector_file]: optional field. 'ngvector_file' is calculated by 'rsem-generate-ngvector'. Having this field is recommended for transcript data.
-number_sample_condition1: the number of samples in condition 1. A condition's samples must be adjacent. The left group of samples are defined as condition 1.
-FDR_rate: false discovery rate.
-output_file: the output file.
+ rsem-control-fdr --help
-The results are written as a matrix with row and column names. The row names are the differentially expressed transcripts'/genes' ids. The column names are 'PPEE', 'PPDE', 'PostFC' and 'RealFC'.
+to get usage information or visit the [rsem-control-fdr documentation
+page](http://deweylab.biostat.wisc.edu/rsem/rsem-control-fdr.html). These
+two scripts can perform DE analysis on either 2 conditions or multiple
+conditions.
-PPEE: posterior probability of being equally expressed.
-PPDE: posterior probability of being differentially expressed.
-PostFC: posterior fold change (condition 1 over condition2).
-RealFC: real fold change (condition 1 over condition2).
-
-To get the above usage information, type
-
- rsem-find-DE
-
-Note: any wrong parameter setting will lead 'rsem-find-DE' to output
-usage information and halt.
+Please note that 'rsem-run-ebseq' and 'rsem-control-fdr' use EBSeq's
+default parameters. For advanced use of EBSeq or information about how
+EBSeq works, please refer to [EBSeq's
+manual](http://www.bioconductor.org/packages/devel/bioc/vignettes/EBSeq/inst/doc/EBSeq_Vignette.pdf).
Questions related to EBSeq should
be sent to <a href="mailto:nleng@wisc.edu">Ning Leng</a>.
We thank earonesty for contributing patches.
+We thank Han Lin for suggesting possible fixes.
+
## <a name="license"></a> License
RSEM is licensed under the [GNU General Public License
projects / rsem.git / blobdiff