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



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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Figure 1: Illustration of purpose and strategy of this work.
Figure 2: Comparison of SLRs and PacBio-CCS on the ERCC sequences.
Figure 3: Comparison of SLRs and PacBio-CCS on human and mouse transcriptomes.
Figure 4: Analysis of novel isoforms revealed by SLR-RNA-seq.
Figure 5: Analysis of distant molecularly associated exon pairs in the human brain transcriptome.
Figure 6: Conservation of distant molecularly associated exon pairs between human and mouse.

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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

Authors and Affiliations



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.

Corresponding author

Correspondence to Michael P Snyder.

Ethics declarations

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.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Tables 1 and 2 and Supplementary Results (PDF 5403 kb)

Supplementary Data Set 1

This is a README describing all the supplementary datasets. (ZIP 324 kb)

Supplementary Data Set 2

Human Molecules per Million measurements for spliced genes. See associated README for file format. (ZIP 231 kb)

Supplementary Data Set 3

Mouse Molecules per Million measurements for spliced genes for both mice combined. See associated README for file format. (ZIP 228 kb)

Supplementary Data Set 4

Mouse Molecules per Million measurements for spliced genes for mouse number 2. See associated README for file format. (ZIP 220 kb)

Supplementary Data Set 5

Human Percent-Spliced-In (Psi) measurements for splice-sites. See associated README for file format. (ZIP 4739 kb)

Supplementary Data Set 6

Mouse Percent-Spliced-In (Psi) measurements for splice-sites for both mice combined. See associated README for file format. (ZIP 2622 kb)

Supplementary Data Set 7

Mouse Percent-Spliced-In (Psi) measurements for splice-sites for mouse number 1. See associated README for file format. (ZIP 2236 kb)

Supplementary Data Set 8

Mouse Percent-Spliced-In (Psi) measurements for splice-sites for mouse number 2. See associated README for file format. (ZIP 1686 kb)

Supplementary Data Set 9

Human Percent-Isoforme (Pi) measurements for spliced genes. See associated README for file format. (ZIP 3023 kb)

Supplementary Data Set 10

Mouse Percent-Isoforme (Pi) measurements for spliced genes for both mice combined. See associated README for file format. (ZIP 1100 kb)

Supplementary Data Set 11

Mouse Percent-Isoforme (Pi) measurements for spliced genes for mouse number 1. See associated README for file format. (ZIP 932 kb)

Supplementary Data Set 12

Mouse Percent-Isoforme (Pi) measurements for spliced genes for mouse number 2. See associated README for file format. (ZIP 695 kb)

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. (ZIP 6 kb)

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. (ZIP 11 kb)

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. (ZIP 1 kb)

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Tilgner, H., Jahanbani, F., Blauwkamp, T. et al. Comprehensive transcriptome analysis using synthetic long-read sequencing reveals molecular co-association of distant splicing events. Nat Biotechnol 33, 736–742 (2015).

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