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Targeted sequencing for gene discovery and quantification using RNA CaptureSeq

Nature Protocols volume 9, pages 989–1009 (2014)Cite this article

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Abstract

RNA sequencing (RNAseq) samples the majority of expressed genes infrequently, owing to the large size, complex splicing and wide dynamic range of eukaryotic transcriptomes. This results in sparse sequencing coverage that can hinder robust isoform assembly and quantification. RNA capture sequencing (CaptureSeq) addresses this challenge by using oligonucleotide probes to capture selected genes or regions of interest for targeted sequencing. Targeted RNAseq provides enhanced coverage for sensitive gene discovery, robust transcript assembly and accurate gene quantification. Here we describe a detailed protocol for all stages of RNA CaptureSeq, from initial probe design considerations and capture of targeted genes to final assembly and quantification of captured transcripts. Initial probe design and final analysis can take less than 1 d, whereas the central experimental capture stage requires ∼7 d.

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Figure 1: Schematic overview of targeted RNAseq.
Figure 2: Schematic overview of targeted RNAseq in three stages: design (red), capture (purple) and analysis (blue).
Figure 3: Designing probes at splice junctions.
Figure 4: Estimated fold enrichment achieved relative to the number of genes targeted.
Figure 5: Example Agilent Bioanalyzer results for pre- and postcapture libraries.
Figure 6: Analysis of ERCC RNA spike-ins to assess the performance of CaptureSeq experiments.
Figure 7: Example of targeted RNAseq expanding the annotation of an lncRNA.

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Acknowledgements

We thank the following funding sources: the Australian National Health and Medical Research Council (Australia Fellowship 631668; to J.S.M., T.R.M. and M.B.C.) and the Queensland State Government (National and International Research Alliance Program; to L.K.N.). We also thank the Institute for Molecular Bioscience core sequencing facility; we thank P. Danoy, J. Jeddeloh (Roche/NimbleGen) and T. Bruxner (Queensland Centre for Medical Genomics) for technical advice and assistance with capture sequencing; and we thank R. Bannen (Roche/NimbleGen) for helping with the design of capture arrays.

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Author notes

  1. Tim R Mercer, Michael B Clark and Joanna Crawford: These authors contributed equally to this work.

Authors and Affiliations

  1. Garvan Institute of Medical Research, Sydney, New South Wales, Australia

    Tim R Mercer, Michael B Clark, Marcel E Dinger & John S Mattick

  2. Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia

    Michael B Clark, Joanna Crawford & Ryan J Taft

  3. Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland, Australia

    Marion E Brunck & Lars K Nielsen

  4. Mater Research Institute—The University of Queensland, Translational Research Institute, Woolloongabba, Queensland, Australia

    Daniel J Gerhardt

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Contributions

T.R.M. and M.E.D. jointly conceived the CaptureSeq strategy. J.C. and M.B.C. designed, optimized and performed all stages of the protocol. T.R.M. and M.B.C. performed the analysis. M.E.B. and D.J.G. contributed to protocol development and optimization. T.R.M., J.C., M.B.C., M.E.B., L.K.N., R.J.T., M.E.D. and J.S.M. prepared the manuscript. L.K.N., R.J.T. and J.S.M. provided funding support.

Corresponding authors

Correspondence to Marcel E Dinger or John S Mattick.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Data

RNA CaptureSeq oligonucleotide sequences. (PDF 351 kb)

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Mercer, T., Clark, M., Crawford, J. et al. Targeted sequencing for gene discovery and quantification using RNA CaptureSeq. Nat Protoc 9, 989–1009 (2014). https://doi.org/10.1038/nprot.2014.058

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