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RNA sequencing: advances, challenges and opportunities

Nature Reviews Genetics volume 12, pages 87–98 (2011)Cite this article

Subjects

Key Points

  • Powered by improving next-generation sequencing capabilities, RNA-seq studies continue to provide knowledge about the quantitative and qualitative aspects of transcriptomes in both prokaryotes and eukaryotes.

  • Advances have been made in areas including the cataloguing of sense and antisense transcripts, alternative splicing events, fused transcripts and transcription initiation sites in physiologically normal and disease settings.

  • RNA-seq studies traditionally rely on sequencing of cDNA libraries that are generated from RNAs through various reverse transcription and sample preparation strategies. Biases and artefacts introduced by these experimental steps may hinder some applications.

  • Recent technological developments, including direct RNA sequencing, may alleviate some of the limitations of current RNA-seq approaches and enable new paths of research into transcriptomics.

  • Although several technological barriers still remain, major advances towards reliable analyses of RNAs from limited cell quantities have recently been achieved, paving the way towards transcriptome profiling at the single-cell level.

Abstract

In the few years since its initial application, massively parallel cDNA sequencing, or RNA-seq, has allowed many advances in the characterization and quantification of transcriptomes. Recently, several developments in RNA-seq methods have provided an even more complete characterization of RNA transcripts. These developments include improvements in transcription start site mapping, strand-specific measurements, gene fusion detection, small RNA characterization and detection of alternative splicing events. Ongoing developments promise further advances in the application of RNA-seq, particularly direct RNA sequencing and approaches that allow RNA quantification from very small amounts of cellular materials.

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Figure 1: RNA-seq for detection of alternative splicing events.
Figure 2: Use of RNA-seq for BCR–ABL fusion gene detection.
Figure 3: Alternative methods for targeted RNA-seq.
Figure 4: Direct RNA sequencing using the Helicos approach.
Figure 5: Emerging technologies for single-cell or low-quantity-cell gene expression profiling.

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Acknowledgements

We apologize to authors whose work could not be cited owing to space constraints. We are grateful to the US National Human Genome Research Institute for their support (grants R01 HG005230 and R44 HG005279).

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    Fatih Ozsolak & Patrice M. Milos

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Glossary

Next generation DNA sequencing

(Often abbreviated to NGS.) Non-Sanger-based high-throughput DNA sequencing technologies. Compared to Sanger sequencing, NGS platforms sequence as many as billions of DNA strands in parallel, yielding substantially more throughput and minimizing the need for the fragment-cloning methods that are often used in Sanger sequencing of genomes.

Semisuppressive PCR

A PCR strategy that aims to reduce primer dimer accumulation by preferentially amplifying longer DNA fragments.

Spike pool

Internal controls added to RNA samples, consisting of RNA elements of known sequence and composition.

Paired-end reads

A strategy involving sequencing of two different regions that are located apart from each other on the same DNA fragment. This strategy provides elevated physical coverage and alleviates several limitations of NGS platforms that arise because of their relatively short read length.

Laser capture microdissection

(Often abbreviated to LCM.) A method allowing cells of interest that are chosen by the operator using a microscope to be specifically captured from heterogeneous tissue samples. The isolated cells can be used for various analyses including of protein and nucleic acid.

Quantitative real-time polymerase chain reaction

A PCR application that enables the measurement of nucleic acid quantities in samples. Nucleic acid of interest is amplified with PCR. The level of the amplified product accumulation during PCR cycles are measured in real time. This data is used to infer starting nucleic acid quantities.

Circulating extracellular nucleic acid

Extracellular DNA or RNA molecules in plasma and serum.

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Ozsolak, F., Milos, P. RNA sequencing: advances, challenges and opportunities. Nat Rev Genet 12, 87–98 (2011). https://doi.org/10.1038/nrg2934

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