SYNOPSIS

DESCRIPTION

reference_name: The name of RSEM references, which should be already generated by 'rsem-prepare-reference' estimated_model_file: This file describes how the RNA-Seq reads will be sequenced given the expression levels. It determines what kind of reads will be simulated (single-end/paired-end, w/o quality score) and includes parameters for fragment length distribution, read start position distribution, sequencing error models, etc. Normally, this file should be learned from real data using 'rsem-calculate-expression'. The file can be found under the 'sample_name.stat' folder with the name of 'sample_name.model' estimated_isoform_results: This file contains expression levels for all isoforms recorded in the reference. It can be learned using 'rsem-calculate-expression' from real data. The corresponding file users want to use is 'sample_name.isoforms.results'. If simulating from user-designed expression profile is desired, start from a learned 'sample_name.isoforms.results' file and only modify the 'TPM' column. The simulator only reads the TPM column. But keeping the file format the same is required. If the RSEM references built are aware of allele-specific transcripts, 'sample_name.alleles.results' should be used instead. theta0: This parameter determines the fraction of reads that are coming from background "noise" (instead of from a transcript). It can also be estimated using 'rsem-calculate-expression' from real data. Users can find it as the first value of the third line of the file 'sample_name.stat/sample_name.theta'. N: The total number of reads to be simulated. If 'rsem-calculate-expression' is executed on a real data set, the total number of reads can be found as the 4th number of the first line of the file 'sample_name.stat/sample_name.cnt'. output_name: Prefix for all output files. --seed seed: Set seed for the random number generator used in simulation. The seed should be a 32-bit unsigned integer. -q: Set it will stop outputting intermediate information.

Outputs:

output_name.sim.isoforms.results, output_name.sim.genes.results: Expression levels estimated by counting where each simulated read comes from. output_name.sim.alleles.results: Allele-specific expression levels estimated by counting where each simulated read comes from.

output_name.fa if single-end without quality score; output_name.fq if single-end with quality score; output_name_1.fa & output_name_2.fa if paired-end without quality score; output_name_1.fq & output_name_2.fq if paired-end with quality score.

Format of the header line: Each simulated read's header line encodes where it comes from. The header line has the format:

{>/@}_rid_dir_sid_pos[_insertL]

{>/@}: Either '>' or '@' must appear. '>' appears if FASTA files are generated and '@' appears if FASTQ files are generated rid: Simulated read's index, numbered from 0 dir: The direction of the simulated read. 0 refers to forward strand ('+') and 1 refers to reverse strand ('-') sid: Represent which transcript this read is simulated from. It ranges between 0 and M, where M is the total number of transcripts. If sid=0, the read is simulated from the background noise. Otherwise, the read is simulated from a transcript with index sid. Transcript sid's transcript name can be found in the 'transcript_id' column of the 'sample_name.isoforms.results' file (at line sid + 1, line 1 is for column names) pos: The start position of the simulated read in strand dir of transcript sid. It is numbered from 0 insertL: Only appear for paired-end reads. It gives the insert length of the simulated read.

Example:

Suppose we want to simulate 50 millon single-end reads with quality scores and use the parameters learned from [Example](#example). In addition, we set theta0 as 0.2 and output_name as 'simulated_reads'. The command is:

rsem-simulate-reads ,/ref/mouse_125/ mmliver_single_quals.stat/mmliver_single_quals.model mmliver_single_quals.isoforms.results 0.2 50000000 simulated_reads