Provided by: rsem_1.3.3+dfsg-2_amd64 bug

NAME

       rsem-prepare-reference - Prepare transcript references for RSEM and optionally build
       BOWTIE/BOWTIE2/STAR/HISAT2(transcriptome) indices.

SYNOPSIS

        rsem-prepare-reference [options] reference_fasta_file(s) reference_name

ARGUMENTS

       reference_fasta_file(s)
           Either a comma-separated list of Multi-FASTA formatted files OR a directory name. If a
           directory name is specified, RSEM will read all files with suffix ".fa" or ".fasta" in
           this directory.  The files should contain either the sequences of transcripts or an
           entire genome, depending on whether the '--gtf' option is used.

       reference name
           The name of the reference used. RSEM will generate several reference-related files
           that are prefixed by this name. This name can contain path information (e.g.
           '/ref/mm9').

OPTIONS

       --gtf <file>
           If this option is on, RSEM assumes that 'reference_fasta_file(s)' contains the
           sequence of a genome, and will extract transcript reference sequences using the gene
           annotations specified in <file>, which should be in GTF format.

           If this and '--gff3' options are off, RSEM will assume 'reference_fasta_file(s)'
           contains the reference transcripts. In this case, RSEM assumes that name of each
           sequence in the Multi-FASTA files is its transcript_id.

           (Default: off)

       --gff3 <file>
           The annotation file is in GFF3 format instead of GTF format. RSEM will first convert
           it to GTF format with the file name 'reference_name.gtf'. Please make sure that
           'reference_name.gtf' does not exist. (Default: off)

       --gff3-RNA-patterns <pattern>
           <pattern> is a comma-separated list of transcript categories, e.g. "mRNA,rRNA". Only
           transcripts that match the <pattern> will be extracted. (Default: "mRNA")

       --gff3-genes-as-transcripts
           This option is designed for untypical organisms, such as viruses, whose GFF3 files
           only contain genes. RSEM will assume each gene as a unique transcript when it converts
           the GFF3 file into GTF format.

       --trusted-sources <sources>
           <sources> is a comma-separated list of trusted sources, e.g. "ENSEMBL,HAVANA". Only
           transcripts coming from these sources will be extracted. If this option is off, all
           sources are accepted. (Default: off)

       --transcript-to-gene-map <file>
           Use information from <file> to map from transcript (isoform) ids to gene ids.  Each
           line of <file> should be of the form:

           gene_id transcript_id

           with the two fields separated by a tab character.

           If you are using a GTF file for the "UCSC Genes" gene set from the UCSC Genome
           Browser, then the "knownIsoforms.txt" file (obtained from the "Downloads" section of
           the UCSC Genome Browser site) is of this format.

           If this option is off, then the mapping of isoforms to genes depends on whether the
           '--gtf' option is specified.  If '--gtf' is specified, then RSEM uses the "gene_id"
           and "transcript_id" attributes in the GTF file.  Otherwise, RSEM assumes that each
           sequence in the reference sequence files is a separate gene.

           (Default: off)

       --allele-to-gene-map <file>
           Use information from <file> to provide gene_id and transcript_id information for each
           allele-specific transcript.  Each line of <file> should be of the form:

           gene_id transcript_id allele_id

           with the fields separated by a tab character.

           This option is designed for quantifying allele-specific expression. It is only valid
           if '--gtf' option is not specified. allele_id should be the sequence names presented
           in the Multi-FASTA-formatted files.

           (Default: off)

       --polyA
           Add poly(A) tails to the end of all reference isoforms. The length of poly(A) tail
           added is specified by '--polyA-length' option. STAR aligner users may not want to use
           this option. (Default: do not add poly(A) tail to any of the isoforms)

       --polyA-length <int>
           The length of the poly(A) tails to be added. (Default: 125)

       --no-polyA-subset <file>
           Only meaningful if '--polyA' is specified. Do not add poly(A) tails to those
           transcripts listed in <file>. <file> is a file containing a list of transcript_ids.
           (Default: off)

       --bowtie
           Build Bowtie indices. (Default: off)

       --bowtie-path <path>
           The path to the Bowtie executables. (Default: the path to Bowtie executables is
           assumed to be in the user's PATH environment variable)

       --bowtie2
           Build Bowtie 2 indices. (Default: off)

       --bowtie2-path <path>
           The path to the Bowtie 2 executables. (Default: the path to Bowtie 2 executables is
           assumed to be in the user's PATH environment variable)

       --star
           Build STAR indices. (Default: off)

       --star-path <path>
           The path to STAR's executable. (Default: the path to STAR executable is assumed to be
           in user's PATH environment variable)

       --star-sjdboverhang <int>
           Length of the genomic sequence around annotated junction. It is only used for STAR to
           build splice junctions database and not needed for Bowtie or Bowtie2. It will be
           passed as the --sjdbOverhang option to STAR. According to STAR's manual, its ideal
           value is max(ReadLength)-1, e.g. for 2x101 paired-end reads, the ideal value is
           101-1=100. In most cases, the default value of 100 will work as well as the ideal
           value. (Default: 100)

       --hisat2-hca
           Build HISAT2 indices on the transcriptome according to Human Cell Atlas (HCA)
           SMART-Seq2 pipeline. (Default: off)

       --hisat2-path <path>
           The path to the HISAT2 executables. (Default: the path to HISAT2 executables is
           assumed to be in the user's PATH environment variable)

       -p/--num-threads <int>
           Number of threads to use for building STAR's genome indices. (Default: 1)

       -q/--quiet
           Suppress the output of logging information. (Default: off)

       -h/--help
           Show help information.

PRIOR-ENHANCED RSEM OPTIONS

       --prep-pRSEM
           A Boolean indicating whether to prepare reference files for pRSEM, including building
           Bowtie indices for a genome and selecting training set isoforms. The index files will
           be used for aligning ChIP-seq reads in prior-enhanced RSEM and the training set
           isoforms will be used for learning prior. A path to Bowtie executables and a
           mappability file in bigWig format are required when this option is on. Currently,
           Bowtie2 is not supported for prior-enhanced RSEM. (Default: off)

       --mappability-bigwig-file <string>
           Full path to a whole-genome mappability file in bigWig format. This file is required
           for running prior-enhanced RSEM. It is used for selecting a training set of isoforms
           for prior-learning. This file can be either downloaded from UCSC Genome Browser or
           generated by GEM (Derrien et al., 2012, PLoS One). (Default: "")

DESCRIPTION

       This program extracts/preprocesses the reference sequences for RSEM and prior-enhanced
       RSEM. It can optionally build Bowtie indices (with '--bowtie' option) and/or Bowtie 2
       indices (with '--bowtie2' option) using their default parameters. It can also optionally
       build STAR indices (with '--star' option) using parameters from ENCODE3's STAR-RSEM
       pipeline. For prior-enhanced RSEM, it can build Bowtie genomic indices and select training
       set isoforms (with options '--prep-pRSEM' and '--mappability-bigwig-file <string>'). If an
       alternative aligner is to be used, indices for that particular aligner can be built from
       either 'reference_name.idx.fa' or 'reference_name.n2g.idx.fa' (see OUTPUT for details).
       This program is used in conjunction with the 'rsem-calculate-expression' program.

OUTPUT

       This program will generate 'reference_name.grp', 'reference_name.ti',
       'reference_name.transcripts.fa', 'reference_name.seq', 'reference_name.chrlist' (if
       '--gtf' is on), 'reference_name.idx.fa', 'reference_name.n2g.idx.fa', optional
       Bowtie/Bowtie 2 index files, and optional STAR index files.

       'reference_name.grp', 'reference_name.ti', 'reference_name.seq', and
       'reference_name.chrlist' are used by RSEM internally.

       'reference_name.transcripts.fa' contains the extracted reference transcripts in Multi-
       FASTA format. Poly(A) tails are not added and it may contain lower case bases in its
       sequences if the corresponding genomic regions are soft-masked.

       'reference_name.idx.fa' and 'reference_name.n2g.idx.fa' are used by aligners to build
       their own indices. In these two files, all sequence bases are converted into upper case.
       In addition, poly(A) tails are added if '--polyA' option is set. The only difference
       between 'reference_name.idx.fa' and 'reference_name.n2g.idx.fa' is that
       'reference_name.n2g.idx.fa' in addition converts all 'N' characters to 'G' characters.
       This conversion is in particular desired for aligners (e.g. Bowtie) that do not allow
       reads to overlap with 'N' characters in the reference sequences. Otherwise,
       'reference_name.idx.fa' should be used to build the aligner's index files. RSEM uses
       'reference_name.idx.fa' to build Bowtie 2 indices and 'reference_name.n2g.idx.fa' to build
       Bowtie indices. For visualizing the transcript-coordinate-based BAM files generated by
       RSEM in IGV, 'reference_name.idx.fa' should be imported as a "genome" (see Visualization
       section in README.md for details).

       If the whole genome is indexed for prior-enhanced RSEM, all the index files will be
       generated with prefix as 'reference_name_prsem'. Selected isoforms for training set are
       listed in the file 'reference_name_prsem.training_tr_crd'

EXAMPLES

       1) Suppose we have mouse RNA-Seq data and want to use the UCSC mm9 version of the mouse
       genome. We have downloaded the UCSC Genes transcript annotations in GTF format (as
       mm9.gtf) using the Table Browser and the knownIsoforms.txt file for mm9 from the UCSC
       Downloads. We also have all chromosome files for mm9 in the directory '/data/mm9'.  We
       want to put the generated reference files under '/ref' with name 'mouse_0'. We do not add
       any poly(A) tails. Please note that GTF files generated from UCSC's Table Browser do not
       contain isoform-gene relationship information.  For the UCSC Genes annotation, this
       information can be obtained from the knownIsoforms.txt file.  Suppose we want to build
       Bowtie indices and Bowtie executables are found in '/sw/bowtie'.

       There are two ways to write the command:

        rsem-prepare-reference --gtf mm9.gtf \
                               --transcript-to-gene-map knownIsoforms.txt \
                               --bowtie \
                               --bowtie-path /sw/bowtie \
                               /data/mm9/chr1.fa,/data/mm9/chr2.fa,...,/data/mm9/chrM.fa \
                               /ref/mouse_0

       OR

        rsem-prepare-reference --gtf mm9.gtf \
                               --transcript-to-gene-map knownIsoforms.txt \
                               --bowtie \
                               --bowtie-path /sw/bowtie \
                               /data/mm9 \
                               /ref/mouse_0

       2) Suppose we also want to build Bowtie 2 indices in the above example and Bowtie 2
       executables are found in '/sw/bowtie2', the command will be:

        rsem-prepare-reference --gtf mm9.gtf \
                               --transcript-to-gene-map knownIsoforms.txt \
                               --bowtie \
                               --bowtie-path /sw/bowtie \
                               --bowtie2 \
                               --bowtie2-path /sw/bowtie2 \
                               /data/mm9 \
                               /ref/mouse_0

       3) Suppose we want to build STAR indices in the above example and save index files under
       '/ref' with name 'mouse_0'. Assuming STAR executable is '/sw/STAR', the command will be:

        rsem-prepare-reference --gtf mm9.gtf \
                               --transcript-to-gene-map knownIsoforms.txt \
                               --star \
                               --star-path /sw/STAR \
                               -p 8 \
                               /data/mm9/chr1.fa,/data/mm9/chr2.fa,...,/data/mm9/chrM.fa \
                               /ref/mouse_0

       OR

        rsem-prepare-reference --gtf mm9.gtf \
                               --transcript-to-gene-map knownIsoforms.txt \
                               --star \
                               --star-path /sw/STAR \
                               -p 8 \
                               /data/mm9
                               /ref/mouse_0

       STAR genome index files will be saved under '/ref/'.

       4) Suppose we want to prepare references for prior-enhanced RSEM in the above example. In
       this scenario, both STAR and Bowtie are required to build genomic indices - STAR for RNA-
       seq reads and Bowtie for ChIP-seq reads. Assuming their executables are under '/sw/STAR'
       and '/sw/Bowtie', respectively. Also, assuming the mappability file for mouse genome is
       '/data/mm9.bigWig'. The command will be:

        rsem-prepare-reference --gtf mm9.gtf \
                               --transcript-to-gene-map knownIsoforms.txt \
                               --star \
                               --star-path /sw/STAR \
                               -p 8 \
                               --prep-pRSEM \
                               --bowtie-path /sw/Bowtie \
                               --mappability-bigwig-file /data/mm9.bigWig \
                               /data/mm9/chr1.fa,/data/mm9/chr2.fa,...,/data/mm9/chrM.fa \
                               /ref/mouse_0

       OR

        rsem-prepare-reference --gtf mm9.gtf \
                               --transcript-to-gene-map knownIsoforms.txt \
                               --star \
                               --star-path /sw/STAR \
                               -p 8 \
                               --prep-pRSEM \
                               --bowtie-path /sw/Bowtie \
                               --mappability-bigwig-file /data/mm9.bigWig \
                               /data/mm9
                               /ref/mouse_0

       Both STAR and Bowtie's index files will be saved under '/ref/'. Bowtie files will have
       name prefix 'mouse_0_prsem'

       5) Suppose we only have transcripts from EST tags stored in 'mm9.fasta' and isoform-gene
       information stored in 'mapping.txt'. We want to add 125bp long poly(A) tails to all
       transcripts. The reference_name is set as 'mouse_125'. In addition, we do not want to
       build Bowtie/Bowtie 2 indices, and will use an alternative aligner to align reads against
       either 'mouse_125.idx.fa' or 'mouse_125.idx.n2g.fa':

        rsem-prepare-reference --transcript-to-gene-map mapping.txt \
                               --polyA
                               mm9.fasta \
                               mouse_125