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Always turn off hyphenation; it makes .\" way too many mistakes in technical documents. .if n .ad l .nh .SH "NAME" rsem\-calculate\-expression .SH "PURPOSE" .IX Header "PURPOSE" Estimate gene and isoform expression from RNA-Seq data. .SH "SYNOPSIS" .IX Header "SYNOPSIS" .Vb 3 \& 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 .Ve .SH "ARGUMENTS" .IX Header "ARGUMENTS" .IP "\fBupstream_read_files(s)\fR" 4 .IX Item "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 \s-1FASTQ\s0 format. If the \-\-no\-qualities option is specified, then \s-1FASTA\s0 format is expected. .IP "\fBdownstream_read_file(s)\fR" 4 .IX Item "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 \s-1FASTQ\s0 format. If the \-\-no\-qualities option is specified, then \s-1FASTA\s0 format is expected. .IP "\fBinput\fR" 4 .IX Item "input" \&\s-1SAM/BAM/CRAM\s0 formatted input file. If \*(L"\-\*(R" is specified for the filename, the input is instead assumed to come from standard input. \s-1RSEM\s0 requires all alignments of the same read group together. For paired-end reads, \s-1RSEM\s0 also requires the two mates of any alignment be adjacent. In addition, \s-1RSEM\s0 does not allow the \s-1SEQ\s0 and \s-1QUAL\s0 fields to be empty. See Description section for how to make input file obey \s-1RSEM\s0's requirements. .IP "\fBreference_name\fR" 4 .IX Item "reference_name" The name of the reference used. The user must have run 'rsem\-prepare\-reference' with this reference_name before running this program. .IP "\fBsample_name\fR" 4 .IX Item "sample_name" The name of the sample analyzed. All output files are prefixed by this name (e.g., sample_name.genes.results) .SH "BASIC OPTIONS" .IX Header "BASIC OPTIONS" .IP "\fB\-\-paired\-end\fR" 4 .IX Item "--paired-end" Input reads are paired-end reads. (Default: off) .IP "\fB\-\-no\-qualities\fR" 4 .IX Item "--no-qualities" Input reads do not contain quality scores. (Default: off) .IP "\fB\-\-strand\-specific\fR" 4 .IX Item "--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 \s-1RSEM\s0 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) .IP "\fB\-p/\-\-num\-threads\fR " 4 .IX Item "-p/--num-threads " Number of threads to use. Both Bowtie/Bowtie2, expression estimation and 'samtools sort' will use this many threads. (Default: 1) .IP "\fB\-\-alignments\fR" 4 .IX Item "--alignments" Input file contains alignments in \s-1SAM/BAM/CRAM\s0 format. The exact file format will be determined automatically. (Default: off) .IP "\fB\-\-fai\fR " 4 .IX Item "--fai " If the header section of input alignment file does not contain reference sequence information, this option should be turned on. is a \s-1FAI\s0 format file containing each reference sequence's name and length. Please refer to the \s-1SAM\s0 official website for the details of \s-1FAI\s0 format. (Default: off) .IP "\fB\-\-bowtie2\fR" 4 .IX Item "--bowtie2" Use Bowtie 2 instead of Bowtie to align reads. Since currently \s-1RSEM\s0 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) .IP "\fB\-\-star\fR" 4 .IX Item "--star" Use \s-1STAR\s0 to align reads. Alignment parameters are from \s-1ENCODE3\s0's STAR-RSEM pipeline. To save computational time and memory resources, \s-1STAR\s0's Output \s-1BAM\s0 file is unsorted. It is stored in \s-1RSEM\s0's temporary directory with name as 'sample_name.bam'. Each \s-1STAR\s0 job will have its own private copy of the genome in memory. (Default: off) .IP "\fB\-\-append\-names\fR" 4 .IX Item "--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) .IP "\fB\-\-seed\fR " 4 .IX Item "--seed " 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 integer. (Default: off) .IP "\fB\-\-single\-cell\-prior\fR" 4 .IX Item "--single-cell-prior" By default, \s-1RSEM\s0 uses \fIDirichlet\fR\|(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 \s-1RSEM\s0 will use Dirichlet(0.1) as the prior which encourage the sparsity of the expression levels. (Default: off) .IP "\fB\-\-calc\-pme\fR" 4 .IX Item "--calc-pme" Run \s-1RSEM\s0's collapsed Gibbs sampler to calculate posterior mean estimates. (Default: off) .IP "\fB\-\-calc\-ci\fR" 4 .IX Item "--calc-ci" Calculate 95% credibility intervals and posterior mean estimates. The credibility level can be changed by setting '\-\-ci\-credibility\-level'. (Default: off) .IP "\fB\-q/\-\-quiet\fR" 4 .IX Item "-q/--quiet" Suppress the output of logging information. (Default: off) .IP "\fB\-h/\-\-help\fR" 4 .IX Item "-h/--help" Show help information. .IP "\fB\-\-version\fR" 4 .IX Item "--version" Show version information. .SH "OUTPUT OPTIONS" .IX Header "OUTPUT OPTIONS" .IP "\fB\-\-sort\-bam\-by\-read\-name\fR" 4 .IX Item "--sort-bam-by-read-name" Sort \s-1BAM\s0 file aligned under transcript coordidate by read name. Setting this option on will produce deterministic maximum likelihood estimations from independent runs. Note that sorting will take long time and lots of memory. (Default: off) .IP "\fB\-\-no\-bam\-output\fR" 4 .IX Item "--no-bam-output" Do not output any \s-1BAM\s0 file. (Default: off) .IP "\fB\-\-sampling\-for\-bam\fR" 4 .IX Item "--sampling-for-bam" When \s-1RSEM\s0 generates a \s-1BAM\s0 file, instead of outputting 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 \*(L"noise\*(R" transcript, which does not appear in the \s-1BAM\s0 file. Only the sampled alignment has a weight of 1. All other alignments have weight 0. If the \*(L"noise\*(R" transcript is sampled, all alignments appeared in the \s-1BAM\s0 file should have weight 0. (Default: off) .IP "\fB\-\-output\-genome\-bam\fR" 4 .IX Item "--output-genome-bam" Generate a \s-1BAM\s0 file, 'sample_name.genome.bam', with alignments mapped to genomic coordinates and annotated with their posterior probabilities. In addition, \s-1RSEM\s0 will call samtools (included in \s-1RSEM\s0 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) .IP "\fB\-\-sort\-bam\-by\-coordinate\fR" 4 .IX Item "--sort-bam-by-coordinate" Sort \s-1RSEM\s0 generated transcript and genome \s-1BAM\s0 files by coordinates and build associated indices. (Default: off) .IP "\fB\-\-sort\-bam\-memory\-per\-thread\fR " 4 .IX Item "--sort-bam-memory-per-thread " Set the maximum memory per thread that can be used by 'samtools sort'. represents the memory and accepts suffices 'K/M/G'. \s-1RSEM\s0 will pass to the '\-m' option of 'samtools sort'. Note that the default used here is different from the default used by samtools. (Default: 1G) .SH "ALIGNER OPTIONS" .IX Header "ALIGNER OPTIONS" .IP "\fB\-\-seed\-length\fR " 4 .IX Item "--seed-length " Seed length used by the read aligner. Providing the correct value is important for \s-1RSEM.\s0 If \s-1RSEM\s0 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) .IP "\fB\-\-phred33\-quals\fR" 4 .IX Item "--phred33-quals" Input quality scores are encoded as Phred+33. (Default: on) .IP "\fB\-\-phred64\-quals\fR" 4 .IX Item "--phred64-quals" Input quality scores are encoded as Phred+64 (default for \s-1GA\s0 Pipeline ver. >= 1.3). (Default: off) .IP "\fB\-\-solexa\-quals\fR" 4 .IX Item "--solexa-quals" Input quality scores are solexa encoded (from \s-1GA\s0 Pipeline ver. < 1.3). (Default: off) .IP "\fB\-\-bowtie\-path\fR " 4 .IX Item "--bowtie-path " The path to the Bowtie executables. (Default: the path to the Bowtie executables is assumed to be in the user's \s-1PATH\s0 environment variable) .IP "\fB\-\-bowtie\-n\fR " 4 .IX Item "--bowtie-n " (Bowtie parameter) max # of mismatches in the seed. (Range: 0\-3, Default: 2) .IP "\fB\-\-bowtie\-e\fR " 4 .IX Item "--bowtie-e " (Bowtie parameter) max sum of mismatch quality scores across the alignment. (Default: 99999999) .IP "\fB\-\-bowtie\-m\fR " 4 .IX Item "--bowtie-m " (Bowtie parameter) suppress all alignments for a read if > valid alignments exist. (Default: 200) .IP "\fB\-\-bowtie\-chunkmbs\fR " 4 .IX Item "--bowtie-chunkmbs " (Bowtie parameter) memory allocated for best first alignment calculation (Default: 0 \- use Bowtie's default) .IP "\fB\-\-bowtie2\-path\fR " 4 .IX Item "--bowtie2-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 \s-1PATH\s0 environment variable) .IP "\fB\-\-bowtie2\-mismatch\-rate\fR " 4 .IX Item "--bowtie2-mismatch-rate " (Bowtie 2 parameter) The maximum mismatch rate allowed. (Default: 0.1) .IP "\fB\-\-bowtie2\-k\fR " 4 .IX Item "--bowtie2-k " (Bowtie 2 parameter) Find up to alignments per read. (Default: 200) .IP "\fB\-\-bowtie2\-sensitivity\-level\fR " 4 .IX Item "--bowtie2-sensitivity-level " (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. must be one of \*(L"very_fast\*(R", \*(L"fast\*(R", \*(L"sensitive\*(R" and \*(L"very_sensitive\*(R". The four candidates correspond to Bowtie 2's \*(L"\-\-very\-fast\*(R", \*(L"\-\-fast\*(R", \*(L"\-\-sensitive\*(R" and \*(L"\-\-very\-sensitive\*(R" options. (Default: \*(L"sensitive\*(R" \- use Bowtie 2's default) .IP "\fB\-\-star\-path\fR " 4 .IX Item "--star-path " The path to \s-1STAR\s0's executable. (Default: the path to \s-1STAR\s0 executable is assumed to be in user's \s-1PATH\s0 environment variable) .IP "\fB\-\-star\-gzipped\-read\-file\fR" 4 .IX Item "--star-gzipped-read-file" (\s-1STAR\s0 parameter) Input read file(s) is compressed by gzip. (Default: off) .IP "\fB\-\-star\-bzipped\-read\-file\fR" 4 .IX Item "--star-bzipped-read-file" (\s-1STAR\s0 parameter) Input read file(s) is compressed by bzip2. (Default: off) .IP "\fB\-\-star\-output\-genome\-bam\fR" 4 .IX Item "--star-output-genome-bam" (\s-1STAR\s0 parameter) Save the \s-1BAM\s0 file from \s-1STAR\s0 alignment under genomic coordinate to 'sample_name.STAR.genome.bam'. This file is \s-1NOT\s0 sorted by genomic coordinate. In this file, according to \s-1STAR\s0's manual, 'paired ends of an alignment are always adjacent, and multiple alignments of a read are adjacent as well'. (Default: off) .SH "ADVANCED OPTIONS" .IX Header "ADVANCED OPTIONS" .IP "\fB\-\-tag\fR " 4 .IX Item "--tag " The name of the optional field used in the \s-1SAM\s0 input for identifying a read with too many valid alignments. The field should have the format :i:, where a bigger than 0 indicates a read with too many alignments. (Default: "") .IP "\fB\-\-forward\-prob\fR " 4 .IX Item "--forward-prob " 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) .IP "\fB\-\-fragment\-length\-min\fR " 4 .IX Item "--fragment-length-min " Minimum read/insert length allowed. This is also the value for the Bowtie/Bowtie2 \-I option. (Default: 1) .IP "\fB\-\-fragment\-length\-max\fR " 4 .IX Item "--fragment-length-max " Maximum read/insert length allowed. This is also the value for the Bowtie/Bowtie 2 \-X option. (Default: 1000) .IP "\fB\-\-fragment\-length\-mean\fR " 4 .IX Item "--fragment-length-mean " (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) .IP "\fB\-\-fragment\-length\-sd\fR " 4 .IX Item "--fragment-length-sd " (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 \fB\-\-fragment\-length\-mean\fR) .IP "\fB\-\-estimate\-rspd\fR" 4 .IX Item "--estimate-rspd" Set this option if you want to estimate the read start position distribution (\s-1RSPD\s0) from data. Otherwise, \s-1RSEM\s0 will use a uniform \s-1RSPD. \s0(Default: off) .IP "\fB\-\-num\-rspd\-bins\fR " 4 .IX Item "--num-rspd-bins " Number of bins in the \s-1RSPD.\s0 Only relevant when '\-\-estimate\-rspd' is specified. Use of the default setting is recommended. (Default: 20) .IP "\fB\-\-gibbs\-burnin\fR " 4 .IX Item "--gibbs-burnin " The number of burn-in rounds for \s-1RSEM\s0's Gibbs sampler. Each round passes over the entire data set once. If \s-1RSEM\s0 can use multiple threads, multiple Gibbs samplers will start at the same time and all samplers share the same burn-in number. (Default: 200) .IP "\fB\-\-gibbs\-number\-of\-samples\fR " 4 .IX Item "--gibbs-number-of-samples " The total number of count vectors \s-1RSEM\s0 will collect from its Gibbs samplers. (Default: 1000) .IP "\fB\-\-gibbs\-sampling\-gap\fR " 4 .IX Item "--gibbs-sampling-gap " The number of rounds between two succinct count vectors \s-1RSEM\s0 collects. If the count vector after round N is collected, the count vector after round N + will also be collected. (Default: 1) .IP "\fB\-\-ci\-credibility\-level\fR " 4 .IX Item "--ci-credibility-level " The credibility level for credibility intervals. (Default: 0.95) .IP "\fB\-\-ci\-memory\fR " 4 .IX Item "--ci-memory " Maximum size (in memory, \s-1MB\s0) of the auxiliary buffer used for computing credibility intervals (\s-1CI\s0). (Default: 1024) .IP "\fB\-\-ci\-number\-of\-samples\-per\-count\-vector\fR " 4 .IX Item "--ci-number-of-samples-per-count-vector " 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) .IP "\fB\-\-keep\-intermediate\-files\fR" 4 .IX Item "--keep-intermediate-files" Keep temporary files generated by \s-1RSEM. RSEM\s0 creates a temporary directory, 'sample_name.temp', into which it puts all intermediate output files. If this directory already exists, \s-1RSEM\s0 overwrites all files generated by previous \s-1RSEM\s0 runs inside of it. By default, after \s-1RSEM\s0 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) .IP "\fB\-\-temporary\-folder\fR " 4 .IX Item "--temporary-folder " Set where to put the temporary files generated by \s-1RSEM.\s0 If the folder specified does not exist, \s-1RSEM\s0 will try to create it. (Default: sample_name.temp) .IP "\fB\-\-time\fR" 4 .IX Item "--time" Output time consumed by each step of \s-1RSEM\s0 to 'sample_name.time'. (Default: off) .SH "DESCRIPTION" .IX Header "DESCRIPTION" In its default mode, this program aligns input reads against a reference transcriptome with Bowtie and calculates expression values using the alignments. \s-1RSEM\s0 assumes the data are single-end reads with quality scores, unless the '\-\-paired\-end' or '\-\-no\-qualities' options are specified. Alternatively, users can use \s-1STAR\s0 to align reads using the '\-\-star' option. \s-1RSEM\s0 has provided options in 'rsem\-prepare\-reference' to prepare \s-1STAR\s0's genome indices. Users may use an alternative aligner by specifying '\-\-alignments', and providing an alignment file in \s-1SAM/BAM/CRAM\s0 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 \s-1ARGUMENTS\s0 section. .PP One simple way to make the alignment file satisfying \s-1RSEM\s0's requirements is to use the 'convert\-sam\-for\-rsem' script. This script accepts \s-1SAM/BAM/CRAM\s0 files as input and outputs a \s-1BAM\s0 file. For example, type the following command to convert a \s-1SAM\s0 file, 'input.sam', to a ready-for-use \s-1BAM\s0 file, 'input_for_rsem.bam': .PP .Vb 1 \& convert\-sam\-for\-rsem input.sam input_for_rsem .Ve .PP For details, please refer to 'convert\-sam\-for\-rsem's documentation page. .SH "NOTES" .IX Header "NOTES" 1. Users must run 'rsem\-prepare\-reference' with the appropriate reference before using this program. .PP 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, \s-1RSEM\s0 will automatically learn a fragment length distribution from the data. .PP 3. Some aligner parameters have default values different from their original settings. .PP 4. With the '\-\-calc\-pme' option, posterior mean estimates will be calculated in addition to maximum likelihood estimates. .PP 5. With the '\-\-calc\-ci' option, 95% credibility intervals and posterior mean estimates will be calculated in addition to maximum likelihood estimates. .PP 6. The temporary directory and all intermediate files will be removed when \s-1RSEM\s0 finishes unless '\-\-keep\-intermediate\-files' is specified. .SH "OUTPUT" .IX Header "OUTPUT" .IP "\fBsample_name.isoforms.results\fR" 4 .IX Item "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: .Sp transcript_id gene_id length effective_length expected_count \s-1TPM FPKM\s0 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] .Sp Fields are separated by the tab character. Fields within \*(L"[]\*(R" are optional. They will not be presented if neither '\-\-calc\-pme' nor \&'\-\-calc\-ci' is set. .Sp \&'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. .Sp \&'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. .Sp \&'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) \s-1RSEM\s0 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. .Sp \&'\s-1TPM\s0' stands for Transcripts Per Million. It is a relative measure of transcript abundance. The sum of all transcripts' \s-1TPM\s0 is 1 million. '\s-1FPKM\s0' 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 .Sp l_bar = \esum_i TPM_i / 10^6 * effective_length_i (i goes through every transcript), .Sp the following equation is hold: .Sp FPKM_i = 10^3 / l_bar * TPM_i. .Sp We can see that the sum of \s-1FPKM\s0 is not a constant across samples. .Sp \&'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. .Sp \&'posterior_mean_count', 'pme_TPM', 'pme_FPKM' are posterior mean estimates calculated by \s-1RSEM\s0'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. .Sp \&'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 \s-1TPM\s0 and \s-1FPKM\s0 values. The bounds are inclusive (i.e. [l, u]). .Sp \&'TPM_coefficient_of_quartile_variation' and \&'FPKM_coefficient_of_quartile_variation' are coefficients of quartile variation (\s-1CQV\s0) for \s-1TPM\s0 and \s-1FPKM\s0 values. \s-1CQV\s0 is a robust way of measuring the ratio between the standard deviation and the mean. It is defined as .Sp \&\s-1CQV\s0 := (Q3 \- Q1) / (Q3 + Q1), .Sp where Q1 and Q3 are the first and third quartiles. .IP "\fBsample_name.genes.results\fR" 4 .IX Item "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: .Sp gene_id transcript_id(s) length effective_length expected_count \s-1TPM FPKM\s0 [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] .Sp Fields are separated by the tab character. Fields within \*(L"[]\*(R" are optional. They will not be presented if neither '\-\-calc\-pme' nor \&'\-\-calc\-ci' is set. .Sp \&'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'). .Sp 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. .IP "\fBsample_name.alleles.results\fR" 4 .IX Item "sample_name.alleles.results" Only generated when the \s-1RSEM\s0 references are built with allele-specific transcripts. .Sp 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: .Sp allele_id transcript_id gene_id length effective_length expected_count \s-1TPM FPKM\s0 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] .Sp Fields are separated by the tab character. Fields within \*(L"[]\*(R" are optional. They will not be presented if neither '\-\-calc\-pme' nor \&'\-\-calc\-ci' is set. .Sp \&'allele_id' is the allele-specific name of this allele-specific transcript. .Sp \&'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. .Sp \&'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. .Sp \&'AlleleIsoPct_from_pme_TPM' and 'AlleleGenePct_from_pme_TPM' have similar meanings. They are calculated based on posterior mean estimates. .Sp 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'. .IP "\fBsample_name.transcript.bam\fR" 4 .IX Item "sample_name.transcript.bam" Only generated when \-\-no\-bam\-output is not specified. .Sp \&'sample_name.transcript.bam' is a BAM-formatted file of read alignments in transcript coordinates. The \s-1MAPQ\s0 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, \s-1RSEM\s0 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 \s-1BAM/SAM\s0 file. .IP "\fBsample_name.transcript.sorted.bam and sample_name.transcript.sorted.bam.bai\fR" 4 .IX Item "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. .Sp \&'sample_name.transcript.sorted.bam' and \&'sample_name.transcript.sorted.bam.bai' are the sorted \s-1BAM\s0 file and indices generated by samtools (included in \s-1RSEM\s0 package). .IP "\fBsample_name.genome.bam\fR" 4 .IX Item "sample_name.genome.bam" Only generated when \-\-no\-bam\-output is not specified and \-\-output\-genome\-bam is specified. .Sp \&'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 \s-1MAPQ\s0 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, \s-1RSEM\s0 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. .IP "\fBsample_name.genome.sorted.bam and sample_name.genome.sorted.bam.bai\fR" 4 .IX Item "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. .Sp \&'sample_name.genome.sorted.bam' and 'sample_name.genome.sorted.bam.bai' are the sorted \s-1BAM\s0 file and indices generated by samtools (included in \s-1RSEM\s0 package). .IP "\fBsample_name.time\fR" 4 .IX Item "sample_name.time" Only generated when \-\-time is specified. .Sp It contains time (in seconds) consumed by aligning reads, estimating expression levels and calculating credibility intervals. .IP "\fBsample_name.stat\fR" 4 .IX Item "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. .Sp \&'sample_name.stat/sample_name.cnt' contains alignment statistics. The format and meanings of each field are described in 'cnt_file_description.txt' under \s-1RSEM\s0 directory. .Sp \&'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 \s-1RSEM\s0 directory. .SH "EXAMPLES" .IX Header "EXAMPLES" Assume the path to the bowtie executables is in the user's \s-1PATH\s0 environment variable. Reference files are under '/ref' with name 'mouse_125'. .PP 1) '/data/mmliver.fq', single-end reads with quality scores. Quality scores are encoded as for '\s-1GA\s0 pipeline version >= 1.3'. We want to use 8 threads and generate a genome \s-1BAM\s0 file. In addition, we want to append gene/transcript names to the result files: .PP .Vb 7 \& rsem\-calculate\-expression \-\-phred64\-quals \e \& \-p 8 \e \& \-\-append\-names \e \& \-\-output\-genome\-bam \e \& /data/mmliver.fq \e \& /ref/mouse_125 \e \& mmliver_single_quals .Ve .PP 2) '/data/mmliver_1.fq' and '/data/mmliver_2.fq', paired-end reads with quality scores. Quality scores are in \s-1SANGER\s0 format. We want to use 8 threads and do not generate a genome \s-1BAM\s0 file: .PP .Vb 6 \& rsem\-calculate\-expression \-p 8 \e \& \-\-paired\-end \e \& /data/mmliver_1.fq \e \& /data/mmliver_2.fq \e \& /ref/mouse_125 \e \& mmliver_paired_end_quals .Ve .PP 3) '/data/mmliver.fa', single-end reads without quality scores. We want to use 8 threads: .PP .Vb 5 \& rsem\-calculate\-expression \-p 8 \e \& \-\-no\-qualities \e \& /data/mmliver.fa \e \& /ref/mouse_125 \e \& mmliver_single_without_quals .Ve .PP 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 \s-1BAM\s0 file, we also want to generate credibility intervals. We allow \s-1RSEM\s0 to use 1GB of memory for \s-1CI\s0 calculation: .PP .Vb 11 \& rsem\-calculate\-expression \-\-bowtie\-path /sw/bowtie \e \& \-\-phred64\-quals \e \& \-\-fragment\-length\-mean 150.0 \e \& \-\-fragment\-length\-sd 35.0 \e \& \-p 8 \e \& \-\-output\-genome\-bam \e \& \-\-calc\-ci \e \& \-\-ci\-memory 1024 \e \& /data/mmliver.fq \e \& /ref/mouse_125 \e \& mmliver_single_quals .Ve .PP 5) '/data/mmliver_paired_end_quals.bam', BAM-formatted alignments for paired-end reads with quality scores. We want to use 8 threads: .PP .Vb 6 \& rsem\-calculate\-expression \-\-paired\-end \e \& \-\-alignments \e \& \-p 8 \e \& /data/mmliver_paired_end_quals.bam \e \& /ref/mouse_125 \e \& mmliver_paired_end_quals .Ve .PP 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 \s-1STAR\s0 to aligned reads and assume \s-1STAR\s0 executable is '/sw/STAR'. Suppose we want to use 8 threads and do not generate a genome \s-1BAM\s0 file: .PP .Vb 9 \& rsem\-calculate\-expression \-\-paired\-end \e \& \-\-star \e \& \-\-star\-path /sw/STAR \e \& \-\-gzipped\-read\-file \e \& \-p 8 \e \& /data/mmliver_1.fq.gz \e \& /data/mmliver_2.fq.gz \e \& /ref/mouse_125 \e \& mmliver_paired_end_quals .Ve