Are small RNAs included in a typical Illumina RNA seq dataset?

Question

[llanoe]

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 (https://cm.jefferson.edu/MINTbase/).

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?

Answer 1

[A. Domingues]

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.

[JC]

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

[llanoe]

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

[ATpoint]

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.