July 27, 2025Open Access

The efficacy and accuracy of ribosomal RNA depletion methods

MAMohammed AhmedUniversity of Liverpool CBCleo L. BishopQueen Mary University of London ABAndrea J. BetancourtUniversity of Liverpool

Key Points

Zymo-Seq showed the highest efficiency in removing ribosomal rna and reducing biases in gene expression.
QIAseq showed the least rRNA removal, leading to significant differential expression biases in analysis.
Three rRNA depletion kits were tested for their performance with the parasitic nematode Strongyloides ratti.
Findings highlight the critical need for validating rRNA depletion methods, particularly for non-model organisms.

Abstract

The depletion of ribosomal RNA (rRNA) is a critical step in RNA-sequence analyses, used to enhance the detection of non-rRNA molecules, such as messenger RNAs and non-coding RNAs. However, the efficiency and potential biases introduced by different rRNA depletion methods remain poorly characterized. Here, we evaluated three commercially available rRNA depletion kits - QIAseq FastSelect, riboPOOL, Zymo-Seq RiboFree - for their performance with the parasitic nematode Strongyloides ratti. We assessed the kits' efficiency in rRNA removal, the recovery of expressed genes and transposable elements, and the detection of spliced leader sequences and genes' operonic organization. Zymo-Seq demonstrated the highest sensitivity and minimal bias in a measure of gene expression, while QIAseq showed the least rRNA depletion and significant differential expression biases. Our findings underscore the importance of empirical validation of rRNA depletion methods, particularly for parasites and non-model organisms, and we suggest that Zymo-Seq as the optimal choice for S. ratti and related nematodes.

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