Small RNA Sequencing Techniques and Applications

Small RNA sequencing encompasses a suite of high-throughput approaches designed to capture and quantify populations of short non-coding RNAs, principally microRNAs, small interfering RNAs and PIWI-interacting RNAs. Central to these methods is the preparation of sequencing libraries, involving adaptor ligation, reverse transcription and amplification steps that aim to preserve the true relative abundance of each RNA species. Early protocols suffered from sequence- and structure-dependent biases introduced during adaptor ligation, prompting the development of randomised and polyethylene glycol-supplemented adaptors, unique molecular identifier tagging and ligation-free workflows. More recently, single-cell small RNA sequencing has emerged, enabling the simultaneous profiling of microRNAs and messenger RNAs in individual cells and uncovering cell-to-cell heterogeneity in gene regulation. Computational pipelines have matured to integrate quality control, alignment, quantification and downstream differential expression analysis, often accommodating novel species discovery and nucleotide-resolution mapping of modifications. Together, these advances have broadened the applications of small RNA sequencing to biomarker discovery in bodily fluids, investigation of host–pathogen interactions, plant and animal stress responses, and the characterization of post-transcriptional regulatory networks in development and disease.

Research from Nature Portfolio

Recent work has demonstrated the power of co-profiling microRNAs and mRNAs within the same single cell, revealing that variability in microRNA expression alone can drive non-genetic heterogeneity and shape downstream gene regulatory networks. By splitting individual cells for parallel microRNA and mRNA library preparation, researchers achieved high reproducibility and confirmed that anti-correlated target expression mirrors microRNA abundance fluctuations. In parallel, an integrated analysis toolkit has been introduced that bundles miRNA, mRNA and circular RNA identification with differential expression and target prediction in a single, multithreaded pipeline. This suite simplifies installation, scales from small to large sample cohorts and brings together established alignment and quantification software to deliver comprehensive small RNA-centric transcriptome analyses.

Small RNA Sequencing Techniques and Applications publication trend

The graph below shows the total number of articles in small rna sequencing techniques and applications across all publications each year (not limited to Nature Index journals).

Technical terms

Adapter ligation bias: Preferential attachment of sequencing adaptors to particular RNA sequences or structures, leading to distorted abundance estimates.

Unique molecular identifier (UMI): A random oligonucleotide tag added to each RNA molecule to distinguish true biological copies from PCR duplicates.

IsomiR: A sequence variant of a microRNA arising from alternative processing or nucleotide additions, which may modulate target specificity.

Ligation-free protocol: A library preparation strategy that avoids enzymatic adaptor ligation to reduce bias and capture modified RNAs.

Single-cell small RNA sequencing (scsRNA-seq): Techniques for profiling small RNA populations at single-cell resolution, often combined with mRNA co-sequencing for integrated analyses.

References

1. Single-cell microRNA-mRNA co-sequencing reveals non-genetic heterogeneity and mechanisms of microRNA regulation. Nature Communications (2019).
2. txtools: an R package facilitating analysis of RNA modifications, structures, and interactions. Nucleic Acids Research (2024).
3. sRNAbench and sRNAtoolbox 2019: intuitive fast small RNA profiling and differential expression. Nucleic Acids Research (2019).
4. Bias in Ligation-Based Small RNA Sequencing Library Construction Is Determined by Adaptor and RNA Structure. PLOS ONE (2015).
5. Biases in small RNA deep sequencing data. Nucleic Acids Research (2013).
6. Addressing Bias in Small RNA Library Preparation for Sequencing: A New Protocol Recovers MicroRNAs that Evade Capture by Current Methods. Frontiers in Genetics (2015).
7. CAP-miRSeq: a comprehensive analysis pipeline for microRNA sequencing data. BMC Genomics (2014).
8. miARma-Seq: a comprehensive tool for miRNA, mRNA and circRNA analysis. Scientific Reports (2016).
9. Biomarker discovery: quantification of microRNAs and other small non-coding RNAs using next generation sequencing. BMC Medical Genomics (2015).