Abstract

We compared quantitative RT-PCR (qRT-PCR), RNA-seq and capture sequencing (CaptureSeq) in terms of their ability to assemble and quantify long noncoding RNAs and novel coding exons across 20 human tissues. CaptureSeq was superior for the detection and quantification of genes with low expression, showed little technical variation and accurately measured differential expression. This approach expands and refines previous annotations and simultaneously generates an expression atlas.

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Acknowledgements

The authors acknowledge the following funding sources: an Australian National Health and Medical Research Council (NHMRC) Australia Fellowship (631668 to J.S.M. and 631542 to M.E.D.), an NHMRC Early Career Fellowship (APP1072662 to M.B.C.), an EMBO Long Term Fellowship (ALTF 864-2013 to M.B.C.), the Queensland State Government (National and International Research Alliance Program to L.K.N.) and an EMBL Interdisciplinary Postdoc (EIPOD) under Marie Curie Actions (COFUND) (to G.B.). The contents of the published material are solely the responsibility of the administering institution, a participating institution or individual authors and do not reflect the views of NHMRC. The authors thank the ENCODE consortium for the provision of data; data were employed in strict accordance with the associated data-release policy. The authors also thank Prof. M. Brown (University of Queensland) for contributions to manuscript preparation.

Author information

Author notes

    • Michael B Clark
    •  & Tim R Mercer

    These authors contributed equally to this work.

Affiliations

  1. Garvan Institute of Medical Research, Sydney, Australia.

    • Michael B Clark
    • , Tim R Mercer
    • , Wendy Y Chen
    • , John S Mattick
    •  & Marcel E Dinger
  2. MRC Functional Genomics Unit, Department of Physiology, Anatomy, and Genetics, University of Oxford, Oxford, UK.

    • Michael B Clark
  3. St Vincents Clinical School, Faculty of Medicine, University of New South Wales, Sydney, Australia.

    • Tim R Mercer
    • , Wendy Y Chen
    • , John S Mattick
    •  & Marcel E Dinger
  4. European Molecular Biology Laboratory–European Bioinformatics Institute (EMBL-EBI), Wellcome Trust Genome Campus, Cambridge, UK.

    • Giovanni Bussotti
    • , Tommaso Leonardi
    •  & Anton J Enright
  5. The University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, Brisbane, Australia.

    • Katelin R Haynes
    • , Kim-Anh Lê Cao
    •  & Gethin P Thomas
  6. Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia.

    • Joanna Crawford
    • , Kim-Anh Lê Cao
    •  & Ryan J Taft
  7. Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Australia.

    • Marion E Brunck
    •  & Lars K Nielsen
  8. Illumina, Inc., San Diego, California, USA.

    • Ryan J Taft
  9. School of Medicine and Health Services, Departments of Integrated Systems Biology and Pediatrics, George Washington University, Washington DC, USA.

    • Ryan J Taft

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Contributions

T.R.M. and M.B.C. conceived the project and designed experiments, with advice from M.E.D. and J.S.M. M.B.C., J.C., M.E.B. and K.R.H. performed RNA-seq and RNA CaptureSeq. T.R.M., M.B.C., T.L. and G.B. performed the bioinformatic analyses. K.-A.L.C. assisted with statistical analyses. W.Y.C. performed PCR validations. T.R.M., M.B.C., M.E.D., L.K.N. and J.S.M. prepared the manuscript. G.P.T., A.J.E., L.K.N., R.J.T., M.E.D. and J.S.M. provided funding.

Competing interests

T.R.M. is a recipient of a Roche Discovery Agreement (2014). M.B.C. has received research support from Roche/Nimblegen for an unrelated research project.

Corresponding authors

Correspondence to John S Mattick or Marcel E Dinger.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–13 and Supplementary Results

Excel files

  1. 1.

    Supplementary Table 1

    PCR primers utilized in this study.

  2. 2.

    Supplementary Table 2

    Capture transcripts containing putative novel coding exons plus associated putative novel coding exons and GENCODE genes.

  3. 3.

    Supplementary Table 3

    Novel coding exon transcript annotations.

Zip files

  1. 1.

    Supplementary Data 1

    Human genome coordinates (hg19) of tiled regions used for lncRNA CaptureSeq experiment.

  2. 2.

    Supplementary Data 2

    Human genome coordinates (hg19) for all captured and assembled noncoding RNAs.

  3. 3.

    Supplementary Data 3

    Human genome coordinates (hg19) for all captured and assembled coding RNAs.

  4. 4.

    Supplementary Data 4

    Human genome coordinates (hg19) for all novel coding RNAs that join LncRNA and coding gene loci.

  5. 5.

    Supplementary Data 5

    Human genome coordinates (hg19) for all novel noncoding RNAs.

  6. 6.

    Supplementary Data 6

    Transcript Annotation file (.gtf) for all assembled transcripts (comprehensive).

  7. 7.

    Supplementary Data 7

    FPKM values for all assembled transcripts (comprehensive).

  8. 8.

    Supplementary Data 8

    Human genome coordinates (hg19) for all putative novel coding exons that were expressed. Exon annotation as per Lindblad-Toh et al. (2011).

  9. 9.

    Supplementary Data 9

    Human genome coordinates (hg19) for all captured and assembled transcripts containing putative novel coding exons.

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DOI

https://doi.org/10.1038/nmeth.3321

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