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Dual RNA-seq of pathogen and host

Nature Reviews Microbiology volume 10, pages 618–630 (2012)Cite this article

Subjects

Key Points

  • During the infection process, the interaction between pathogen and host results in large-scale changes in gene expression within both organisms.

  • Owing to technical limitations, previous transcriptomic studies using probe- and tag-based approaches often required the separation of the interacting host and pathogen. However, the development of a new technology, RNA sequencing (RNA-seq), now promises to allow the analysis of both partners simultaneously.

  • This dual RNA-seq approach is not without difficulties, owing to the very different nature of bacterial and eukaryotic cells, and especially the properties of the RNAs in these two different domains of life.

  • There are various technical considerations required for the successful determination of the host and pathogen transcriptomes. An analysis of these considerations has led to the proposal that the dual RNA-seq approach is currently becoming feasible and will probably become the gold standard for host–pathogen transcriptomics in the future.

Abstract

A comprehensive understanding of host–pathogen interactions requires a knowledge of the associated gene expression changes in both the pathogen and the host. Traditional, probe-dependent approaches using microarrays or reverse transcription PCR typically require the pathogen and host cells to be physically separated before gene expression analysis. However, the development of the probe-independent RNA sequencing (RNA-seq) approach has begun to revolutionize transcriptomics. Here, we assess the feasibility of taking transcriptomics one step further by performing 'dual RNA-seq', in which gene expression changes in both the pathogen and the host are analysed simultaneously.

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Figure 1: Fundamental differences between probe-dependent and probe-independent approaches to gene expression analysis.
Figure 2: A paradigm shift in parallel host–pathogen transcriptomics.
Figure 3: Estimation of the minimal sequencing depth required for dual host–pathogen RNA-seq.

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Acknowledgements

The authors acknowledge support from the German Research Foundation (DFG) Priority Program SPP1258 (DFG grant Vo875/4-2) and from the German Ministry of Education and Research (BMBF) (grant 01GS0806). A.J.W. is the recipient of an Elite Advancement Ph.D. stipend from the Universität Bayern e.V., Germany.

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Authors and Affiliations

  1. Institute for Molecular Infection Biology, University of Würzburg, D-97080, Germany

    Alexander J. Westermann, Stanislaw A. Gorski & Jörg Vogel

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  1. Alexander J. Westermann
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  2. Stanislaw A. Gorski
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Correspondence to Jörg Vogel.

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Supplementary information

Supplementary Table S1

Selection of (pro- and eukaryotic) organisms to which full-genome RNA-seq has been applied to (PDF 367 kb)

Supplementary Table S2

Steady increase in sequencing depth from early to current RNA-seq studies in both bacteria and mammals (PDF 331 kb)

Supplementary Table S3

Selection of reported values of copy numbers of different bacterial species per host cell (PDF 244 kb)

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Glossary

Pathogen-associated molecular patterns

(PAMPs).General small molecular motifs that are present on microorganisms and engage host innate immune receptors, in particular Toll-like receptors. Examples of PAMPs include lipopolysaccharide, peptidoglycan and flagellin.

Tiling arrays

DNA microarray chips on which probe sequences are tiled (overlapping) and comprise a subset of, or the whole, genome at high resolution.

Small non-coding RNA

A short transcript (∼50–500 nucleotides) that regulates gene expression in bacteria, often by base-pairing with mRNAs.

microRNA

A short (∼22 nucleotide) processed RNA that guides post-transcriptional repression of mRNAs in animals and plants.

Long non-coding RNAs

Heterogeneous non-coding RNAs (>200 nucleotides) that lack protein-coding capability and are found in eukaryotes.

Small nuclear RNAs

Short RNAs that are involved in precursor mRNA processing.

Small nucleolar RNAs

RNAs that typically guide ribose methylation and pseudouridylation in other RNA molecules.

Bar-code sequences

Short unique sequence tags (∼4–6 nucleotides) that are incorporated into cDNA fragments and used to tag a specific sequence as belonging to a particular cDNA library.

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Westermann, A., Gorski, S. & Vogel, J. Dual RNA-seq of pathogen and host. Nat Rev Microbiol 10, 618–630 (2012). https://doi.org/10.1038/nrmicro2852

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