Managing abundant molecules in small RNA sequencing.

Introduction

Small RNA sequencing enables the profiling of diverse RNA species under 200 nucleotides in length, including microRNA (miRNAs), transfer RNA (tRNA), Y RNA, PIWI-interacting RNA (piRNA), and others. However, a recurring challenge in many datasets is the overrepresentation of a few highly abundant RNA species. Most notably, tRNA, tRNA-derived fragments, Y RNA and a small number of highly expressed miRNAs frequently dominate the sequencing output, often obscuring the detection of rare and biologically relevant small RNAs.

This skewed distribution reflects real biological abundance but creates a bottleneck for discovery and quantification of low-abundance species. In this post, we review the current literature supporting this phenomenon across species and tissues, and discuss effective strategies, particularly blocking oligonucleotides, to mitigate this issue and increase library diversity.

tRNA-derived fragments and Y RNAs dominate read counts

The sheer number of tRNA and tRNA fragments in the cells is well-established. It has been shown that more than 90% of small RNA molecules in yeast and mammalian cells are derived from tRNAs, highlighting their overwhelming abundance¹. Other studies have confirmed that tRNA derived fragments from nucleus and organelles appear across virtually all tissues in eukaryotes, including plants, often increasing in response to stress or cellular changes^2,3.

In the extracellular space the situation is similar. A comprehensive study looking at plasma, urine, and saliva from healthy individuals found that Y RNA fragments comprised ~ 63% of mapped reads in plasma, while tRNA fragments dominated in urine and saliva⁴. This makes Y RNA the most abundant non-coding RNA species in plasma small RNA libraries. Y RNA is also present at high levels in the extracellular vesicles of several cell lines⁵. Finally, it has been demonstrated that fragments from glycine and glutamic acid tRNAs are stable and enriched in human extracellular vesicles⁶. Some library preparations like the NEXTFLEX™ Small RNA Sequencing kit v4 provide the option of blocking human, mouse and rat tRNA and Y RNA species during library prep. However, for other organisms, available options are limited.

A few miRNAs dominate expression profiles

It has been observed repeatedly that a small number of miRNAs account for the majority of total miRNA reads, even in complex tissues and across diverse organisms. This distribution is a biologically conserved feature and can skew the interpretability of sequencing results.

For example, miR-486-5p and miR-451a, two erythroid-specific miRNAs, can represent up to 80% of total miRNA reads in human blood-derived libraries⁷. In mouse brain and testis, miR-124, miR-9, and miR-34c are consistently dominant, reducing the representation of other miRNAs^8,9.

In Drosophila, miR-8, bantam, miR-184, and miR-1 have been identified as dominating the miRNA landscape across development. Very similar to Aedes aegypti mosquitoes, where miR-1, miR-184, and miR-275 have been shown to be the most abundant across life stages^10,11.  And in C. elegans, let-7, miR-58, and miR-1 are the leading miRNAs in adults¹².

Finally, in plants similar observations have been reported. For example, in the panicle of rice miR-156 is highly expressed and in maize, miR‑166 and miR‑168 are consistently among the highest‑expressed miRNAs in seeds^13,14. 

Blocking strategies: reducing dominant RNA species in libraries

To counteract the overabundance of specific RNA species, Revvity offers the possibility of incorporating custom blocking oligonucleotides. These oligos are designed to hybridize to the 5' or 3' end of abundant RNAs (miRNA, tRNA, rRNA fragments, Y RNA) in the first step of the NEXTFLEX Small RNA Sequencing kit workflow.  Hybridization prevents 3’ ligation and therefore the target RNA cannot be amplified downstream. Using this strategy, miR-486-5p and miR-451a in human blood libraries were reduced up to 99%, with up to 33% gain in detectability of other miRNAs⁷.

Conclusion

The dominance of tRNA fragments and a handful of miRNAs is biologically real and poses serious challenges to library complexity and the discovery of low-abundance regulatory RNAs. Fortunately, oligonucleotide-based blocking strategies offer a powerful and flexible way to reduce this impact.

By incorporating blockers in the small RNA sequencing workflow, researchers can increase effective library complexity, improve quantification of low-abundance miRNAs and even customize the sequencing output for tissue- or physiological condition-specific discovery.