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Comprehensive transcriptome analysis using synthetic long-read sequencing reveals molecular co-association of distant splicing events

Nature Biotechnology volume 33, pages 736742 (2015) | Download Citation



Alternative splicing shapes mammalian transcriptomes, with many RNA molecules undergoing multiple distant alternative splicing events. Comprehensive transcriptome analysis, including analysis of exon co-association in the same molecule, requires deep, long-read sequencing. Here we introduce an RNA sequencing method, synthetic long-read RNA sequencing (SLR-RNA-seq), in which small pools (≤1,000 molecules/pool, ≤1 molecule/gene for most genes) of full-length cDNAs are amplified, fragmented and short-read-sequenced. We demonstrate that these RNA sequences reconstructed from the short reads from each of the pools are mostly close to full length and contain few insertion and deletion errors. We report many previously undescribed isoforms (human brain: 13,800 affected genes, 14.5% of molecules; mouse brain 8,600 genes, 18% of molecules) and up to 165 human distant molecularly associated exon pairs (dMAPs) and distant molecularly and mutually exclusive pairs (dMEPs). Of 16 associated pairs detected in the mouse brain, 9 are conserved in human. Our results indicate conserved mechanisms that can produce distant but phased features on transcript and proteome isoforms.

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We thank N. Spies and F.A. Bava for a thorough reading of this manuscript and valuable comments and S. Shringarpure, V. Kuleshov, C.S. Foo and H. Tang for valuable comments on statistics. We thank A. Brunet for providing mice and S. Munro for valuable comments on this manuscript. We also thank the Genetics Bioinformatics Service Center at Stanford for providing a well-working computing cluster. M.R. is paid by grant 12-131829 from the Danish Council for Independent Research. This work was supported by grant 5U01HL10739304 (to M.S. as co-PI), 1P50HG007735-01 (to M.S. as co-PI) and 5P01GM09913004 (to M.S.).

Author information

Author notes

    • Hagen Tilgner
    •  & Fereshteh Jahanbani

    These authors contributed equally to this work.


  1. Department of Genetics, Stanford University, Stanford, California, USA.

    • Hagen Tilgner
    • , Fereshteh Jahanbani
    • , Itamar Harel
    • , Carlos D Bustamante
    • , Morten Rasmussen
    •  & Michael P Snyder
  2. Illumina Inc., San Francisco, California, USA.

    • Tim Blauwkamp
    • , Ali Moshrefi
    • , Erich Jaeger
    •  & Feng Chen


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H.T., T.B., F.C. and M.P.S. devised the project. F.J., T.B., E.J., A.M. and M.R. carried out experiments. I.H. euthanized mice and extracted brains. H.T. carried out computational analysis. C.D.B. and M.P.S. supervised the project and provided financial support. H.T. wrote the first version of the manuscript. H.T., F.J., M.R. and M.P.S. wrote the final version of the manuscript with contributions from the other authors.

Competing interests

A. Moshrefi, E. Jaeger and F. Chen are employees of Illumina. T. Blauwkamp is a former employee of Illumina. M. Snyder is on the scientific advisory board of Personalis, GenapSys and AxioMx. C. Bustamante is a founder of Identify Genomics. He is also on the Scientific Advisory Board of Identify, Etalon, Personalis and He is a former member of the advisory board member of InVitae. None of these organizations played a role in the design or conduct of the work presented here.

Corresponding author

Correspondence to Michael P Snyder.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–8 and Supplementary Tables 1 and 2 and Supplementary Results

Zip files

  1. 1.

    Supplementary Data Set 1

    This is a README describing all the supplementary datasets.

  2. 2.

    Supplementary Data Set 2

    Human Molecules per Million measurements for spliced genes. See associated README for file format.

  3. 3.

    Supplementary Data Set 3

    Mouse Molecules per Million measurements for spliced genes for both mice combined. See associated README for file format.

  4. 4.

    Supplementary Data Set 4

    Mouse Molecules per Million measurements for spliced genes for mouse number 2. See associated README for file format.

  5. 5.

    Supplementary Data Set 5

    Human Percent-Spliced-In (Psi) measurements for splice-sites. See associated README for file format.

  6. 6.

    Supplementary Data Set 6

    Mouse Percent-Spliced-In (Psi) measurements for splice-sites for both mice combined. See associated README for file format.

  7. 7.

    Supplementary Data Set 7

    Mouse Percent-Spliced-In (Psi) measurements for splice-sites for mouse number 1. See associated README for file format.

  8. 8.

    Supplementary Data Set 8

    Mouse Percent-Spliced-In (Psi) measurements for splice-sites for mouse number 2. See associated README for file format.

  9. 9.

    Supplementary Data Set 9

    Human Percent-Isoforme (Pi) measurements for spliced genes. See associated README for file format.

  10. 10.

    Supplementary Data Set 10

    Mouse Percent-Isoforme (Pi) measurements for spliced genes for both mice combined. See associated README for file format.

  11. 11.

    Supplementary Data Set 11

    Mouse Percent-Isoforme (Pi) measurements for spliced genes for mouse number 1. See associated README for file format.

  12. 12.

    Supplementary Data Set 12

    Mouse Percent-Isoforme (Pi) measurements for spliced genes for mouse number 2. See associated README for file format.

  13. 13.

    Supplementary Data Set 13

    Human "distant Molecularly Associated Pairs" (dMAPs) of exons and "distant Molecularly and Mutually Exclusive Pairs" (dMEPs) of exons using only human brain RNA. See associated README for file format.

  14. 14.

    Supplementary Data Set 14

    Human "distant Molecularly Associated Pairs" (dMAPs) of exons and "distant Molecularly and Mutually Exclusive Pairs" (dMEPs) of exons using human brain RNA and a variety of previously published long read RNA-datasets (Tilgner et al, GGG, 2013; Sharon et al, Nature Biotechnology, 2013; Tilgner et al, PNAS, 2014). See associated README for file format.

  15. 15.

    Supplementary Data Set 15

    Mouse "distant Molecularly Associated Pairs" (dMAPs) of exons and "distant Molecularly and Mutually Exclusive Pairs" (dMEPs) of exons using only mouse brain RNA. See associated README for file format.

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