rsem-calculate-expression --help

NAME
    rsem-calculate-expression

SYNOPSIS
     rsem-calculate-expression [options] upstream_read_file(s) reference_name sample_name
     rsem-calculate-expression [options] --paired-end upstream_read_file(s) downstream_read_file(s) reference_name sample_name
     rsem-calculate-expression [options] --alignments [--paired-end] input reference_name sample_name

ARGUMENTS
    upstream_read_files(s)
        Comma-separated list of files containing single-end reads or upstream
        reads for paired-end data. By default, these files are assumed to be
        in FASTQ format. If the --no-qualities option is specified, then FASTA
        format is expected.

    downstream_read_file(s)
        Comma-separated list of files containing downstream reads which are
        paired with the upstream reads. By default, these files are assumed to
        be in FASTQ format. If the --no-qualities option is specified, then
        FASTA format is expected.

    input
        SAM/BAM/CRAM formatted input file. If "-" is specified for the
        filename, the input is instead assumed to come from standard input.
        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. In addition, RSEM does not allow the SEQ and QUAL fields to
        be empty. See Description section for how to make input file obey
        RSEM's requirements.

    reference_name
        The name of the reference used. The user must have run
        'rsem-prepare-reference' with this reference_name before running this
        program.

    sample_name
        The name of the sample analyzed. All output files are prefixed by this
        name (e.g., sample_name.genes.results)

BASIC OPTIONS
    --paired-end
        Input reads are paired-end reads. (Default: off)

    --no-qualities
        Input reads do not contain quality scores. (Default: off)

    --strand-specific
        The RNA-Seq protocol used to generate the reads is strand specific,
        i.e., all (upstream) reads are derived from the forward strand. This
        option is equivalent to --forward-prob=1.0. With this option set, if
        RSEM runs the Bowtie/Bowtie 2 aligner, the '--norc' Bowtie/Bowtie 2
        option will be used, which disables alignment to the reverse strand of
        transcripts. (Default: off)

    -p/--num-threads <int>
        Number of threads to use. Both Bowtie/Bowtie2, expression estimation
        and 'samtools sort' will use this many threads. (Default: 1)

    --alignments
        Input file contains alignments in SAM/BAM/CRAM format. The exact file
        format will be determined automatically. (Default: off)

    --fai <file>
        RSEM reads header information from input by default. If this option is
        on, header information is read from the specified file. For the format
        of the file, please see SAM official website. (Default: off)

    --bowtie2
        Use Bowtie 2 instead of Bowtie to align reads. Since currently RSEM
        does not handle indel, local and discordant alignments, the Bowtie2
        parameters are set in a way to avoid those alignments. In particular,
        we use options '--sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1
        --score-min L,0,-0.1' by default. The last parameter of '--score-min',
        '-0.1', is the negative of maximum mismatch rate. This rate can be set
        by option '--bowtie2-mismatch-rate'. If reads are paired-end, we
        additionally use options '--no-mixed' and '--no-discordant'. (Default:
        off)

    --star
        Use STAR to align reads. Alignment parameters are from ENCODE3's
        STAR-RSEM pipeline. To save computational time and memory resources,
        STAR's Output BAM file is unsorted. It is stored in RSEM's temporary
        directory with name as 'sample_name.bam'. Each STAR job will have its
        own private copy of the genome in memory. (Default: off)

    --append-names
        If gene_name/transcript_name is available, append it to the end of
        gene_id/transcript_id (separated by '_') in files
        'sample_name.isoforms.results' and 'sample_name.genes.results'.
        (Default: off)

    --seed <uint32>
        Set the seed for the random number generators used in calculating
        posterior mean estimates and credibility intervals. The seed must be a
        non-negative 32 bit interger. (Default: off)

    --single-cell-prior
        By default, RSEM uses Dirichlet(1) as the prior to calculate posterior
        mean estimates and credibility intervals. However, much less genes are
        expressed in single cell RNA-Seq data. Thus, if you want to compute
        posterior mean estimates and/or credibility intervals and you have
        single-cell RNA-Seq data, you are recommended to turn on this option.
        Then RSEM will use Dirichlet(0.1) as the prior which encourage the
        sparsity of the expression levels. (Default: off)

    --calc-pme
        Run RSEM's collapsed Gibbs sampler to calculate posterior mean
        estimates. (Default: off)

    --calc-ci
        Calculate 95% credibility intervals and posterior mean estimates. The
        credibility level can be changed by setting '--ci-credibility-level'.
        (Default: off)

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

    -h/--help
        Show help information.

    --version
        Show version information.

OUTPUT OPTIONS
    --sort-bam-by-read-name
        Sort BAM file aligned under transcript coordidate by read name.
        Setting this option on will produce determinstic maximum likelihood
        estimations from independent runs. Note that sorting will take long
        time and lots of memory. (Default: off)

    --no-bam-output
        Do not output any BAM file. (Default: off)

    --sampling-for-bam
        When RSEM generates a BAM file, instead of outputing all alignments a
        read has with their posterior probabilities, one alignment is sampled
        according to the posterior probabilities. The sampling procedure
        includes the alignment to the "noise" transcript, which does not
        appear in the BAM file. Only the sampled alignment has a weight of 1.
        All other alignments have weight 0. If the "noise" transcript is
        sampled, all alignments appeared in the BAM file should have weight 0.
        (Default: off)

    --output-genome-bam
        Generate a BAM file, 'sample_name.genome.bam', with alignments mapped
        to genomic coordinates and annotated with their posterior
        probabilities. In addition, RSEM will call samtools (included in RSEM
        package) to sort and index the bam file.
        'sample_name.genome.sorted.bam' and
        'sample_name.genome.sorted.bam.bai' will be generated. (Default: off)

    --sort-bam-by-coordinate
        Sort RSEM generated transcript and genome BAM files by coordinates and
        build associated indices. (Default: off)

    --sort-bam-memory <string>
        Set the maximum memory per thread that can be used by 'samtools sort'.
        <string> represents the memory and accepts suffices 'K/M/G'. RSEM will
        pass <string> to the '-m' option of 'samtools sort'. Note that the
        default used here is different from the default used by samtools.
        (Default: 1G)

ALIGNER OPTIONS
    --seed-length <int>
        Seed length used by the read aligner. Providing the correct value is
        important for RSEM. If RSEM runs Bowtie, it uses this value for
        Bowtie's seed length parameter. Any read with its or at least one of
        its mates' (for paired-end reads) length less than this value will be
        ignored. If the references are not added poly(A) tails, the minimum
        allowed value is 5, otherwise, the minimum allowed value is 25. Note
        that this script will only check if the value >= 5 and give a warning
        message if the value < 25 but >= 5. (Default: 25)

    --phred33-quals
        Input quality scores are encoded as Phred+33. (Default: on)

    --phred64-quals
        Input quality scores are encoded as Phred+64 (default for GA Pipeline
        ver. >= 1.3). (Default: off)

    --solexa-quals
        Input quality scores are solexa encoded (from GA Pipeline ver. < 1.3).
        (Default: off)

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

    --bowtie-n <int>
        (Bowtie parameter) max # of mismatches in the seed. (Range: 0-3,
        Default: 2)

    --bowtie-e <int>
        (Bowtie parameter) max sum of mismatch quality scores across the
        alignment. (Default: 99999999)

    --bowtie-m <int>
        (Bowtie parameter) suppress all alignments for a read if > <int> valid
        alignments exist. (Default: 200)

    --bowtie-chunkmbs <int>
        (Bowtie parameter) memory allocated for best first alignment
        calculation (Default: 0 - use Bowtie's default)

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

    --bowtie2-mismatch-rate <double>
        (Bowtie 2 parameter) The maximum mismatch rate allowed. (Default: 0.1)

    --bowtie2-k <int>
        (Bowtie 2 parameter) Find up to <int> alignments per read. (Default:
        200)

    --bowtie2-sensitivity-level <string>
        (Bowtie 2 parameter) Set Bowtie 2's preset options in --end-to-end
        mode. This option controls how hard Bowtie 2 tries to find alignments.
        <string> must be one of "very_fast", "fast", "sensitive" and
        "very_sensitive". The four candidates correspond to Bowtie 2's
        "--very-fast", "--fast", "--sensitive" and "--very-sensitive" options.
        (Default: "sensitive" - use Bowtie 2's default)

    --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-gzipped-read-file
        (STAR parameter) Input read file(s) is compressed by gzip. (Default:
        off)

    --star-bzipped-read-file
        (STAR parameter) Input read file(s) is compressed by bzip2. (Default:
        off)

    --star-output-genome-bam
        (STAR parameter) Save the BAM file from STAR alignment under genomic
        coordinate to 'sample_name.STAR.genome.bam'. This file is NOT sorted
        by genomic coordinate. In this file, according to STAR's manual,
        'paired ends of an alignment are always adjacent, and multiple
        alignments of a read are adjacent as well'. (Default: off)

ADVANCED OPTIONS
    --tag <string>
        The name of the optional field used in the SAM input for identifying a
        read with too many valid alignments. The field should have the format
        <tagName>:i:<value>, where a <value> bigger than 0 indicates a read
        with too many alignments. (Default: "")

    --forward-prob <double>
        Probability of generating a read from the forward strand of a
        transcript. Set to 1 for a strand-specific protocol where all
        (upstream) reads are derived from the forward strand, 0 for a
        strand-specific protocol where all (upstream) read are derived from
        the reverse strand, or 0.5 for a non-strand-specific protocol.
        (Default: 0.5)

    --fragment-length-min <int>
        Minimum read/insert length allowed. This is also the value for the
        Bowtie/Bowtie2 -I option. (Default: 1)

    --fragment-length-max <int>
        Maximum read/insert length allowed. This is also the value for the
        Bowtie/Bowtie 2 -X option. (Default: 1000)

    --fragment-length-mean <double>
        (single-end data only) The mean of the fragment length distribution,
        which is assumed to be a Gaussian. (Default: -1, which disables use of
        the fragment length distribution)

    --fragment-length-sd <double>
        (single-end data only) The standard deviation of the fragment length
        distribution, which is assumed to be a Gaussian. (Default: 0, which
        assumes that all fragments are of the same length, given by the
        rounded value of --fragment-length-mean)

    --estimate-rspd
        Set this option if you want to estimate the read start position
        distribution (RSPD) from data. Otherwise, RSEM will use a uniform
        RSPD. (Default: off)

    --num-rspd-bins <int>
        Number of bins in the RSPD. Only relevant when '--estimate-rspd' is
        specified. Use of the default setting is recommended. (Default: 20)

    --gibbs-burnin <int>
        The number of burn-in rounds for RSEM's Gibbs sampler. Each round
        passes over the entire data set once. If RSEM can use multiple
        threads, multiple Gibbs samplers will start at the same time and all
        samplers share the same burn-in number. (Default: 200)

    --gibbs-number-of-samples <int>
        The total number of count vectors RSEM will collect from its Gibbs
        samplers. (Default: 1000)

    --gibbs-sampling-gap <int>
        The number of rounds between two succinct count vectors RSEM collects.
        If the count vector after round N is collected, the count vector after
        round N + <int> will also be collected. (Default: 1)

    --ci-credibility-level <double>
        The credibility level for credibility intervals. (Default: 0.95)

    --ci-memory <int>
        Maximum size (in memory, MB) of the auxiliary buffer used for
        computing credibility intervals (CI). (Default: 1024)

    --ci-number-of-samples-per-count-vector <int>
        The number of read generating probability vectors sampled per sampled
        count vector. The crebility intervals are calculated by first sampling
        P(C | D) and then sampling P(Theta | C) for each sampled count vector.
        This option controls how many Theta vectors are sampled per sampled
        count vector. (Default: 50)

    --keep-intermediate-files
        Keep temporary files generated by RSEM. RSEM creates a temporary
        directory, 'sample_name.temp', into which it puts all intermediate
        output files. If this directory already exists, RSEM overwrites all
        files generated by previous RSEM runs inside of it. By default, after
        RSEM finishes, the temporary directory is deleted. Set this option to
        prevent the deletion of this directory and the intermediate files
        inside of it. (Default: off)

    --temporary-folder <string>
        Set where to put the temporary files generated by RSEM. If the folder
        specified does not exist, RSEM will try to create it. (Default:
        sample_name.temp)

    --time
        Output time consumed by each step of RSEM to 'sample_name.time'.
        (Default: off)

DESCRIPTION
    In its default mode, this program aligns input reads against a reference
    transcriptome with Bowtie and calculates expression values using the
    alignments. RSEM assumes the data are single-end reads with quality
    scores, unless the '--paired-end' or '--no-qualities' options are
    specified. Alternatively, users can use STAR to align reads using the
    '--star' option. RSEM has provided options in 'rsem-prepare-reference' to
    prepare STAR's genome indices. Users may use an alternative aligner by
    specifying '--alignments', and providing an alignment file in SAM/BAM/CRAM
    format. However, users should make sure that they align against the
    indices generated by 'rsem-prepare-reference' and the alignment file
    satisfies the requirements mentioned in ARGUMENTS section.

    One simple way to make the alignment file satisfying RSEM's requirements
    is to use the 'convert-sam-for-rsem' script. This script accepts
    SAM/BAM/CRAM files as input and outputs a BAM file. For example, type the
    following command to convert a SAM file, 'input.sam', to a ready-for-use
    BAM file, 'input_for_rsem.bam':

      convert-sam-for-rsem input.sam input_for_rsem

    For details, please refer to 'convert-sam-for-rsem's documentation page.

NOTES
    1. Users must run 'rsem-prepare-reference' with the appropriate reference
    before using this program.

    2. For single-end data, it is strongly recommended that the user provide
    the fragment length distribution parameters (--fragment-length-mean and
    --fragment-length-sd). For paired-end data, RSEM will automatically learn
    a fragment length distribution from the data.

    3. Some aligner parameters have default values different from their
    original settings.

    4. With the '--calc-pme' option, posterior mean estimates will be
    calculated in addition to maximum likelihood estimates.

    5. With the '--calc-ci' option, 95% credibility intervals and posterior
    mean estimates will be calculated in addition to maximum likelihood
    estimates.

    6. The temporary directory and all intermediate files will be removed when
    RSEM finishes unless '--keep-intermediate-files' is specified.

OUTPUT
    sample_name.isoforms.results
        File containing isoform level expression estimates. The first line
        contains column names separated by the tab character. The format of
        each line in the rest of this file is:

        transcript_id gene_id length effective_length expected_count TPM FPKM
        IsoPct [posterior_mean_count posterior_standard_deviation_of_count
        pme_TPM pme_FPKM IsoPct_from_pme_TPM TPM_ci_lower_bound
        TPM_ci_upper_bound TPM_coefficient_of_quartile_variation
        FPKM_ci_lower_bound FPKM_ci_upper_bound
        FPKM_coefficient_of_quartile_variation]

        Fields are separated by the tab character. Fields within "[]" are
        optional. They will not be presented if neither '--calc-pme' nor
        '--calc-ci' is set.

        'transcript_id' is the transcript name of this transcript. 'gene_id'
        is the gene name of the gene which this transcript belongs to (denote
        this gene as its parent gene). If no gene information is provided,
        'gene_id' and 'transcript_id' are the same.

        'length' is this transcript's sequence length (poly(A) tail is not
        counted). 'effective_length' counts only the positions that can
        generate a valid fragment. If no poly(A) tail is added,
        'effective_length' is equal to transcript length - mean fragment
        length + 1. If one transcript's effective length is less than 1, this
        transcript's both effective length and abundance estimates are set to
        0.

        'expected_count' is the sum of the posterior probability of each read
        comes from this transcript over all reads. Because 1) each read
        aligning to this transcript has a probability of being generated from
        background noise; 2) RSEM may filter some alignable low quality reads,
        the sum of expected counts for all transcript are generally less than
        the total number of reads aligned.

        'TPM' stands for Transcripts Per Million. It is a relative measure of
        transcript abundance. The sum of all transcripts' TPM is 1 million.
        'FPKM' stands for Fragments Per Kilobase of transcript per Million
        mapped reads. It is another relative measure of transcript abundance.
        If we define l_bar be the mean transcript length in a sample, which
        can be calculated as

        l_bar = \sum_i TPM_i / 10^6 * effective_length_i (i goes through every
        transcript),

        the following equation is hold:

        FPKM_i = 10^3 / l_bar * TPM_i.

        We can see that the sum of FPKM is not a constant across samples.

        'IsoPct' stands for isoform percentage. It is the percentage of this
        transcript's abandunce over its parent gene's abandunce. If its parent
        gene has only one isoform or the gene information is not provided,
        this field will be set to 100.

        'posterior_mean_count', 'pme_TPM', 'pme_FPKM' are posterior mean
        estimates calculated by RSEM's Gibbs sampler.
        'posterior_standard_deviation_of_count' is the posterior standard
        deviation of counts. 'IsoPct_from_pme_TPM' is the isoform percentage
        calculated from 'pme_TPM' values.

        'TPM_ci_lower_bound', 'TPM_ci_upper_bound', 'FPKM_ci_lower_bound' and
        'FPKM_ci_upper_bound' are lower(l) and upper(u) bounds of 95%
        credibility intervals for TPM and FPKM values. The bounds are
        inclusive (i.e. [l, u]).

        'TPM_coefficient_of_quartile_variation' and
        'FPKM_coefficient_of_quartile_variation' are coefficients of quartile
        variation (CQV) for TPM and FPKM values. CQV is a robust way of
        measuring the ratio between the standard deviation and the mean. It is
        defined as

        CQV := (Q3 - Q1) / (Q3 + Q1),

        where Q1 and Q3 are the first and third quartiles.

    sample_name.genes.results
        File containing gene level expression estimates. The first line
        contains column names separated by the tab character. The format of
        each line in the rest of this file is:

        gene_id transcript_id(s) length effective_length expected_count TPM
        FPKM [posterior_mean_count posterior_standard_deviation_of_count
        pme_TPM pme_FPKM TPM_ci_lower_bound TPM_ci_upper_bound
        TPM_coefficient_of_quartile_variation FPKM_ci_lower_bound
        FPKM_ci_upper_bound FPKM_coefficient_of_quartile_variation]

        Fields are separated by the tab character. Fields within "[]" are
        optional. They will not be presented if neither '--calc-pme' nor
        '--calc-ci' is set.

        'transcript_id(s)' is a comma-separated list of transcript_ids
        belonging to this gene. If no gene information is provided, 'gene_id'
        and 'transcript_id(s)' are identical (the 'transcript_id').

        A gene's 'length' and 'effective_length' are defined as the weighted
        average of its transcripts' lengths and effective lengths (weighted by
        'IsoPct'). A gene's abundance estimates are just the sum of its
        transcripts' abundance estimates.

    sample_name.alleles.results
        Only generated when the RSEM references are built with allele-specific
        transcripts.

        This file contains allele level expression estimates for
        allele-specific expression calculation. The first line contains column
        names separated by the tab character. The format of each line in the
        rest of this file is:

        allele_id transcript_id gene_id length effective_length expected_count
        TPM FPKM AlleleIsoPct AlleleGenePct [posterior_mean_count
        posterior_standard_deviation_of_count pme_TPM pme_FPKM
        AlleleIsoPct_from_pme_TPM AlleleGenePct_from_pme_TPM
        TPM_ci_lower_bound TPM_ci_upper_bound
        TPM_coefficient_of_quartile_variation FPKM_ci_lower_bound
        FPKM_ci_upper_bound FPKM_coefficient_of_quartile_variation]

        Fields are separated by the tab character. Fields within "[]" are
        optional. They will not be presented if neither '--calc-pme' nor
        '--calc-ci' is set.

        'allele_id' is the allele-specific name of this allele-specific
        transcript.

        'AlleleIsoPct' stands for allele-specific percentage on isoform level.
        It is the percentage of this allele-specific transcript's abundance
        over its parent transcript's abundance. If its parent transcript has
        only one allele variant form, this field will be set to 100.

        'AlleleGenePct' stands for allele-specific percentage on gene level.
        It is the percentage of this allele-specific transcript's abundance
        over its parent gene's abundance.

        'AlleleIsoPct_from_pme_TPM' and 'AlleleGenePct_from_pme_TPM' have
        similar meanings. They are calculated based on posterior mean
        estimates.

        Please note that if this file is present, the fields 'length' and
        'effective_length' in 'sample_name.isoforms.results' should be
        interpreted similarly as the corresponding definitions in
        'sample_name.genes.results'.

    sample_name.transcript.bam
        Only generated when --no-bam-output is not specified.

        'sample_name.transcript.bam' is a BAM-formatted file of read
        alignments in transcript coordinates. The MAPQ field of each alignment
        is set to min(100, floor(-10 * log10(1.0 - w) + 0.5)), where w is the
        posterior probability of that alignment being the true mapping of a
        read. In addition, RSEM pads a new tag ZW:f:value, where value is a
        single precision floating number representing the posterior
        probability. Because this file contains all alignment lines produced
        by bowtie or user-specified aligners, it can also be used as a
        replacement of the aligner generated BAM/SAM file.

    sample_name.transcript.sorted.bam and
    sample_name.transcript.sorted.bam.bai
        Only generated when --no-bam-output is not specified and
        --sort-bam-by-coordinate is specified.

        'sample_name.transcript.sorted.bam' and
        'sample_name.transcript.sorted.bam.bai' are the sorted BAM file and
        indices generated by samtools (included in RSEM package).

    sample_name.genome.bam
        Only generated when --no-bam-output is not specified and
        --output-genome-bam is specified.

        'sample_name.genome.bam' is a BAM-formatted file of read alignments in
        genomic coordinates. Alignments of reads that have identical genomic
        coordinates (i.e., alignments to different isoforms that share the
        same genomic region) are collapsed into one alignment. The MAPQ field
        of each alignment is set to min(100, floor(-10 * log10(1.0 - w) +
        0.5)), where w is the posterior probability of that alignment being
        the true mapping of a read. In addition, RSEM pads a new tag
        ZW:f:value, where value is a single precision floating number
        representing the posterior probability. If an alignment is spliced, a
        XS:A:value tag is also added, where value is either '+' or '-'
        indicating the strand of the transcript it aligns to.

    sample_name.genome.sorted.bam and
    sample_name.genome.sorted.bam.bai
        Only generated when --no-bam-output is not specified, and
        --sort-bam-by-coordinate and --output-genome-bam are specified.

        'sample_name.genome.sorted.bam' and
        'sample_name.genome.sorted.bam.bai' are the sorted BAM file and
        indices generated by samtools (included in RSEM package).

    sample_name.time
        Only generated when --time is specified.

        It contains time (in seconds) consumed by aligning reads, estimating
        expression levels and calculating credibility intervals.

    sample_name.stat
        This is a folder instead of a file. All model related statistics are
        stored in this folder. Use 'rsem-plot-model' can generate plots using
        this folder.

        'sample_name.stat/sample_name.cnt' contains alignment statistics. The
        format and meanings of each field are described in
        'cnt_file_description.txt' under RSEM directory.

        'sample_name.stat/sample_name.model' stores RNA-Seq model parameters
        learned from the data. The format and meanings of each filed of this
        file are described in 'model_file_description.txt' under RSEM
        directory.

EXAMPLES
    Assume the path to the bowtie executables is in the user's PATH
    environment variable. Reference files are under '/ref' with name
    'mouse_125'.

    1) '/data/mmliver.fq', single-end reads with quality scores. Quality
    scores are encoded as for 'GA pipeline version >= 1.3'. We want to use 8
    threads and generate a genome BAM file. In addition, we want to append
    gene/transcript names to the result files:

     rsem-calculate-expression --phred64-quals \
                               -p 8 \
                               --append-names \
                               --output-genome-bam \
                               /data/mmliver.fq \
                               /ref/mouse_125 \
                               mmliver_single_quals

    2) '/data/mmliver_1.fq' and '/data/mmliver_2.fq', paired-end reads with
    quality scores. Quality scores are in SANGER format. We want to use 8
    threads and do not generate a genome BAM file:

     rsem-calculate-expression -p 8 \
                               --paired-end \
                               /data/mmliver_1.fq \
                               /data/mmliver_2.fq \
                               /ref/mouse_125 \
                               mmliver_paired_end_quals

    3) '/data/mmliver.fa', single-end reads without quality scores. We want to
    use 8 threads:

     rsem-calculate-expression -p 8 \
                               --no-qualities \
                               /data/mmliver.fa \
                               /ref/mouse_125 \
                               mmliver_single_without_quals

    4) Data are the same as 1). This time we assume the bowtie executables are
    under '/sw/bowtie'. We want to take a fragment length distribution into
    consideration. We set the fragment length mean to 150 and the standard
    deviation to 35. In addition to a BAM file, we also want to generate
    credibility intervals. We allow RSEM to use 1GB of memory for CI
    calculation:

     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 \
                               --ci-memory 1024 \
                               /data/mmliver.fq \
                               /ref/mouse_125 \
                               mmliver_single_quals

    5) '/data/mmliver_paired_end_quals.bam', BAM-formatted alignments for
    paired-end reads with quality scores. We want to use 8 threads:

     rsem-calculate-expression --paired-end \
                               --alignments \
                               -p 8 \
                               /data/mmliver_paired_end_quals.bam \
                               /ref/mouse_125 \
                               mmliver_paired_end_quals

    6) '/data/mmliver_1.fq.gz' and '/data/mmliver_2.fq.gz', paired-end reads
    with quality scores and read files are compressed by gzip. We want to use
    STAR to aligned reads and assume STAR executable is '/sw/STAR'. Suppose we
    want to use 8 threads and do not generate a genome BAM file:

     rsem-calculate-expression --paired-end \
                               --star \
                               --star-path /sw/STAR \
                               --gzipped-read-file \
                               -p 8 \
                               /data/mmliver_1.fq.gz \
                               /data/mmliver_2.fq.gz \
                               /ref/mouse_125 \
                               mmliver_paired_end_quals