RSEM-SIMULATE-READS(1)

📦
version 1.3.3 · 5 versions
, in ubuntu noble, ubuntu resolute

💖 RSEM-SIMULATE-READS 💖

okay bestie, gather round, because this little icon of a command is from the User Commands crew and it is literally here to Simulate RNA-Seq data ("reads") for a given model and a RSEM reference transcript collection. no cap. 🧬✨

✨ SYNOPSIS ✨

rsem-simulate-reads reference_name estimated_model_file estimated_isoform_results theta0 N output_name [--seed seed] [-q]

🌸 DESCRIPTION 🌸

🎀 Parameters 🎀

  • reference_name — the actual name of your RSEM reference, and yes she should already exist, because 'rsem-prepare-reference' is the one who made her 💅
  • estimated_model_file — this file is the whole vibe-setter, fr fr: it describes how the RNA-Seq reads will be sequenced given the expression levels. It decides what kind of reads get simulated (single-end or paired-end, with or without quality score) and it's packing the parameters for fragment length distribution, read start position distribution, sequencing error models, etc. Normally you learn it from real data with 'rsem-calculate-expression', and you'll find it hanging out under the 'sample_name.stat' folder with the name 'sample_name.model' 🫶
  • estimated_isoform_results — this one holds expression levels for every isoform recorded in the reference. Learn it from real data via 'rsem-calculate-expression'; the file you want is 'sample_name.isoforms.results'. Wanna simulate from a user-designed expression profile? then 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, periodt. And if your RSEM references were built aware of allele-specific transcripts, use 'sample_name.alleles.results' instead 👑
  • theta0 — this parameter sets the fraction of reads coming from background "noise" (as opposed to from a transcript). It can also be estimated from real data with 'rsem-calculate-expression', and you'll spot it as the first value of the third line of 'sample_name.stat/sample_name.theta' 🔍
  • N — the total number of reads to be simulated. If 'rsem-calculate-expression' got run on a real data set, the total number of reads shows up as the 4th number of the first line of 'sample_name.stat/sample_name.cnt' 🔢
  • output_name — prefix for all output files. that's her, that's the name 💖
  • --seed seed — set the seed for the random number generator used in simulation. The seed should be a 32-bit unsigned integer ⭐
  • -q — set it and she stops outputting intermediate information. quiet queen behavior 🤫

💅 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

and then the sequence files, depending on the flavor:

  • 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 🔥

Every simulated read's header line encodes where it comes from, and the format is:

{>/@}_rid_dir_sid_pos[_insertL]

breaking it all the way down:

  • {>/@} — either '>' or '@' must appear. '>' appears if FASTA files are generated and '@' appears if FASTQ files are generated
  • rid — the 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 — 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 appears for paired-end reads. It gives the insert length of the simulated read

🦋 Example 🦋

say we wanna simulate 50 million single-end reads with quality scores, and use the parameters learned from [Example](#example). also we're setting 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
lowkey obsessed with how much this one command can do. go forth and simulate, bestie 🌈

this has been rsem-simulate-reads, version 1.3.3+dfsg, dated April 2024 — and that's the whole page, slay 💜🎀🦋✨

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