RNA sequencing: the teenage years

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Over the past decade, RNA sequencing (RNA-seq) has become an indispensable tool for transcriptome-wide analysis of differential gene expression and differential splicing of mRNAs. However, as next-generation sequencing technologies have developed, so too has RNA-seq. Now, RNA-seq methods are available for studying many different aspects of RNA biology, including single-cell gene expression, translation (the translatome) and RNA structure (the structurome). Exciting new applications are being explored, such as spatial transcriptomics (spatialomics). Together with new long-read and direct RNA-seq technologies and better computational tools for data analysis, innovations in RNA-seq are contributing to a fuller understanding of RNA biology, from questions such as when and where transcription occurs to the folding and intermolecular interactions that govern RNA function.

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Fig. 1: Short-read, long-read and direct RNA-seq technologies and workflows.
Fig. 2: RNA-seq data analysis workflow for differential gene expression.
Fig. 3: The key concepts of single-cell and spatial RNA-seq.
Fig. 4: The key concepts of nascent RNA and translatome analysis.
Fig. 5: The key concepts of translatome analysis.
Fig. 6: The key concepts of RNA structure and RNA–protein interaction analysis.


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We thank J. Marioni and J. Ule for their valuable comments on the manuscript.

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J.H., R.S. and M.G. researched the literature. J.H. and R.S. discussed the content and wrote and edited the article.

Correspondence to James Hadfield.

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Supplementary information

Supplementary Information


Differential gene expression

(DGE). The analysis methods that together allow users to determine the quantitative changes in expression levels between experimental groups.

Read depth

The total number of sequencing reads obtained for a sample. This should not be confused with coverage, or sequencing depth, in genome sequencing, which refers to how many times individual nucleotides are sequenced.


Sequencing technologies that generate reads of up to 500 bp, more commonly 100–300 bp, that represent fragmented or degraded mRNAs.


Sequencing technologies that generate reads of over 1,000 bp that represent either full-length or near-full-length mRNAs.

Direct RNA sequencing

(dRNA-seq). Sequencing technologies that generate reads by directly sequencing RNA without modification or reverse transcription, usually with the aim of sequencing full-length or near-full-length mRNAs.

Multi-mapped reads

Sequencing reads from homologous regions of the transcriptome that cannot be unambiguously mapped to the transcriptome or genome.

Synthetic long reads

A method for generating long reads from multiple short reads by assembly.

Unique molecular identifiers

(UMIs). Short sequences or barcodes usually added during RNA sequencing (RNA-seq) library preparation (but also by direct RNA ligation), before amplification, that mark a sequence read as coming from a specific starting molecule. The approach is used to reduce the quantitative biases of RNA-seq and is particularly useful in low-input or single-cell experiments.

Read length

The length of the individual sequencing reads, which is usually 50–150 bp for short-read RNA sequencing.


A measure of the proportion of transcripts present in the sample that are detected. It is affected by sample handling, library preparation, sequencing and computational biases.


A measure of the proportion of differentially expressed transcripts that are correctly identified. It is affected by sample handling, library preparation, sequencing and computational biases.

Tag read

A read that is unique to a transcript, usually from the 3΄ end of mRNA, for differential gene expression analysis, or the 5΄ end, for analysis of transcription start sites and promoters.

Duplication rates

The frequencies at which sequencing reads for an RNA sequencing (RNA-seq) sample map to the same location in the transcriptome. In RNA-seq libraries, duplication rates can seem high for some transcripts because they are present at wildly different levels in the sample. Highly expressed genes will have high duplication rates, while low expressors may have minimal duplication. RNA-seq presents a particular challenge, as much of the duplication may be genuine signal from highly expressed transcripts, while some may be attributable to amplification and sequencing biases.

Single-end sequencing

Short-read sequencing performed from one end of the cDNA fragment, commonly used for differential gene expression experiments, due to its low cost.

Paired-end sequencing

Short-read sequencing performed from both ends of the cDNA fragment, often used for differential gene expression experiments, where maximum sensitivity to splicing is required because more bases of the individual cDNAs will be sequenced.

Biological replicates

Parallel measurements of biologically distinct samples, such as tissue from three subjects, that capture natural biological variation, which may itself be either a subject of study or a source of noise. By contrast, technical replicates are repeated measurements of the same sample — for example, the same tissue processed three times.

Expression matrix

Matrix of values capturing the essential data for a differential-expression RNA-seq experiment. Rows are RNA features, such as genes or transcripts, with one column per sequenced sample. Values are generally counts of the number of reads associated with each RNA feature; these may be estimated for isoform features and are often transformed via normalization before subsequent analysis.

Spike-in control

A pool of exogenous nucleic acids added at known concentration to a sample before processing. They are usually synthetic RNAs pre-pooled at varying concentrations and used to monitor reaction efficiency and to identify methodological bias and false-negative results.


Transcriptome analysis methods that preserve the spatial information of individual transcripts within a given sample, usually a tissue section.

Nascent RNA

RNA that has just been transcribed, as opposed to RNA that has been processed and transported to the cytoplasm.


(4 sU). A thio-substituted nucleoside not naturally found in eukaryotic mRNAs, which is easily incorporated into nucleic acids and is used in nascent RNA analysis.


The complete set of proteins translated from mRNA in a cell, tissue or organism.


The complete set of secondary and tertiary RNA structures in a cell, tissue or organism.


The complete set of molecular interactions in a cell, tissue or organism, including RNA–RNA or RNA–protein interactions.

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