Computational way to distinguish between polyA and total RNA bulk RNA-seq

Question

[predeus]

Hi all,

I was wondering if you know any published method that could allow one to identify if the bulk RNA-seq experiment in question was poly-A selected or done using "total RNA" (ribo-Zero + random hexamer priming or something similar).

As always, would appreciate any suggestions.

-- Alexander

Answer 1

[jared.andrews07]

From a practical perspective, total RNA libraries are typically sequenced more deeply given the greater number and variety of RNAs present. 100M+ reads per sample isn't uncommon, which would be pretty overkill for polyA-selected libraries.

There are also papers with polyA(-) gene lists that you could check. Like this one.

[predeus]

Oh that's great, the list is perfect. I can run a quick evaluation and see the ratio of polyA(-) to total genes, should be a pretty easy call. Thank you.

Answer 2

[GenoMax]

Used GPT to generate a concise answer. You can look for the differences noted below. Looking for rRNA (and the fraction of the data that is rRNA) may be quick method.

Total RNA-seq includes all RNA species within a sample, which means it retains ribosomal RNA (rRNA), along with messenger RNA (mRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), and other non-coding RNAs. In contrast, poly(A)-selected RNA-seq specifically enriches for polyadenylated mRNAs, typically depleting rRNA and other non-polyadenylated RNAs.

Here are the key differences:

A. rRNA Presence:

Total RNA-seq: Contains a large proportion of rRNA, often requiring additional depletion (e.g., Ribo-Zero) to avoid sequencing bias.
Poly(A)-selected RNA-seq: Depletes rRNA inherently by selecting polyadenylated mRNA.

B. Diverse RNA Representation:

Total RNA-seq: Captures a broader spectrum of RNA species, including non-polyadenylated RNAs such as histone mRNAs, lncRNAs, and rRNAs.
Poly(A)-selected RNA-seq: Focuses mainly on coding mRNAs and some polyadenylated non-coding RNAs.