Question: Are small RNAs included in a typical Illumina RNA seq dataset?
gravatar for llanoe
28 days ago by
llanoe0 wrote:

I'm starting to learn RNA seq analysis. I have a paired-end, stranded RNA dataset sequenced with Illumina TruSeq; it includes RNA from a human cell line in multiple conditions. I've used Galaxy to do differential mRNA expression analysis successfully.

The lab I'm working with is now curious about differential expression of little-studied group of small RNAs called tRNA-derived fragments, or tRFs for short. tRFs are ~20 nucleotides in length and there are ~23,000 known human tRFs, and these sequences are listed in an online database (

Before I invest time in trying to learn to do this analysis, I'm trying to figure out whether these small RNAs would even be present in the raw data I have.

Are small RNAs like this typically removed during the processing of the biological sample? or can I expect all the different types of RNA to be present in the raw data?

rna-seq • 130 views
ADD COMMENTlink modified 28 days ago by A. Domingues1.8k • written 28 days ago by llanoe0
gravatar for A. Domingues
28 days ago by
A. Domingues1.8k
Mainz, Germany
A. Domingues1.8k wrote:

Short answer Probably not.

Long answer You will need to check with whomever prepared the libraries, but judging from the description alone

I have a paired-end, stranded RNA dataset sequenced with Illumina TruSeq

I would say it is highly unlikely that there are any small RNAs there for a few reasons:

  • I don't know anyone who does PE sequencing for small RNA because it is a waste of reads (money)
  • Crucially, TrueSep is a "cost-effective solution for analysis of the coding transcriptome, with minimal hands-on time", meaning there is a poly-A capture step and I don't think tRNA-derived fragments have them. But please check because this is crucial point.
  • sometimes in library prep there is a size selection step which could exclude these very small RNAs. Once again check with the person responsible for this.
ADD COMMENTlink written 28 days ago by A. Domingues1.8k

I agree on this, you could see only precursor sequences in your library for such molecules.

ADD REPLYlink written 28 days ago by JC7.6k

Makes sense to me. I will check with the person who processed the samples to confirm. Thanks for your help!

ADD REPLYlink written 28 days ago by llanoe0

Unlikely to have smallRNAs in it. First you need a special RNA extraction kit to capture the small RNAs and second, the typical size selection step for standard RNA-seq isolates fragments of somewhat 200 or more bp, small RNAs would be excluded.

ADD REPLYlink modified 27 days ago • written 27 days ago by ATpoint14k
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