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Targeted RNA sequencing reveals the deep complexity of the human transcriptome

Abstract

Transcriptomic analyses have revealed an unexpected complexity to the human transcriptome, whose breadth and depth exceeds current RNA sequencing capability1,2,3,4. Using tiling arrays to target and sequence select portions of the transcriptome, we identify and characterize unannotated transcripts whose rare or transient expression is below the detection limits of conventional sequencing approaches. We use the unprecedented depth of coverage afforded by this technique to reach the deepest limits of the human transcriptome, exposing widespread, regulated and remarkably complex noncoding transcription in intergenic regions, as well as unannotated exons and splicing patterns in even intensively studied protein-coding loci such as p53 and HOX. The data also show that intermittent sequenced reads observed in conventional RNA sequencing data sets, previously dismissed as noise, are in fact indicative of unassembled rare transcripts. Collectively, these results reveal the range, depth and complexity of a human transcriptome that is far from fully characterized.

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Figure 1: Circle plots illustrating the prevalence and complexity of captured transcripts at genic (a) and intergenic (b) loci.
Figure 2: Resolution of unannotated p53 isoforms.
Figure 3: Identification of unannotated exon variants and rare intergenic noncoding RNAs by targeted RNA capture and sequencing.

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Acknowledgements

The authors would like to thank M. Garber for his design of the arrays and constructive contribution to the manuscript; M. Koziol and K. Thomas for preparing cDNA and sequencing libraries; T. Albert, T. Arnold, J. Affourtit, B. Dessany, T. Jarvie, D. Green and T. Millard provided sequencing support. The authors would like to thank the following funding sources: Human Frontiers Science Program (to T.R.M.); Queensland Government Department of Employment, Economic Development and Innovation Smart Futures Fellowship (to M.E.D.); Australian Research Council/University of Queensland co-sponsored Federation Fellowship (FF0561986; to J.S.M.); Australian National Health and Medical Research Council Australia Fellowship (631668; to J.S.M.) and Career Development Award (CDA631542; to M.E.D.); Damon Runyon-Rachleff, Searle, Smith Family Foundation and Richard Merkin Foundation Scholar (to J.L.R.); and US National Institutes of Health (1DP2OD00667-01; to J.L.R. and C.T.).

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Authors

Contributions

T.R.M., J.A.J., J.S.M. and J.L.R. designed the experiments. D.J.G. performed array capture, quality assessments and supported the sequencing teams. J.C. performed RT-PCR. M.E.D., T.R.M. and C.T. performed alignment, transcript assembly and analysis. T.R.M., M.E.D., J.A.J., J.S.M. and J.L.R. wrote the manuscript.

Corresponding authors

Correspondence to Jeffrey A Jeddeloh, John S Mattick or John L Rinn.

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Competing interests

T.R.M., M.E.D., C.T., J.C., J.S.M. and J.L.R. declare no competing financial interests. D.J.G. and J.A.J are employees of Roche NimbleGen, Inc.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Results (PDF 1707 kb)

Supplementary Tables 1-4

Summary of library sequencing and alignment employed within study. (XLSX 219 kb)

Supplementary Data

Size and genome corrdinates of probed regions (ZIP 3238 kb)

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Mercer, T., Gerhardt, D., Dinger, M. et al. Targeted RNA sequencing reveals the deep complexity of the human transcriptome. Nat Biotechnol 30, 99–104 (2012). https://doi.org/10.1038/nbt.2024

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