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Mapping and quantifying mammalian transcriptomes by RNA-Seq

Abstract

We have mapped and quantified mouse transcriptomes by deeply sequencing them and recording how frequently each gene is represented in the sequence sample (RNA-Seq). This provides a digital measure of the presence and prevalence of transcripts from known and previously unknown genes. We report reference measurements composed of 41–52 million mapped 25-base-pair reads for poly(A)-selected RNA from adult mouse brain, liver and skeletal muscle tissues. We used RNA standards to quantify transcript prevalence and to test the linear range of transcript detection, which spanned five orders of magnitude. Although >90% of uniquely mapped reads fell within known exons, the remaining data suggest new and revised gene models, including changed or additional promoters, exons and 3′ untranscribed regions, as well as new candidate microRNA precursors. RNA splice events, which are not readily measured by standard gene expression microarray or serial analysis of gene expression methods, were detected directly by mapping splice-crossing sequence reads. We observed 1.45 × 105 distinct splices, and alternative splices were prominent, with 3,500 different genes expressing one or more alternate internal splices.

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Figure 1: Outline of RNA-Seq procedure.
Figure 2: Reproducibility, linearity and sensitivity.
Figure 3: Enhanced Read Analysis of Gene Expression (ERANGE) and the allocation of multireads.
Figure 4: Candidate new and revised exons identified by the RNAFAR algorithm.
Figure 5: Candidate microRNA precursor.

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Acknowledgements

This work was supported by The Beckman Foundation, The Simons Foundation and US National Institutes of Health (NIH) grant U54 HG004576 to B.W. and R. Myers. A.M. was supported by an NIH training grant. The authors especially thank D. Trout and B. King for professional data handling and G. Schroth, I. Khrebtukova and S. Luo, of Illumina, for exchanges of preliminary data and protocols under development. M. Liu and J.L. Riechmann, along with others from the laboratories of B. Wold, R. Myers, J. Allman and P. Sternberg, are gratefully acknowledged for many helpful discussions, as are R. Myers and S. Mango for manuscript assistance.

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Correspondence to Barbara Wold.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Tables 1–4 and Supplementary Methods (PDF 500 kb)

Supplementary Software (ZIP 94 kb)

Supplementary Dataset 1

Intermediate and final RPKM values for mouse brain. (TXT 1429 kb)

Supplementary Dataset 2

Intermediate and final RPKM values for mouse liver. (TXT 1423 kb)

Supplementary Dataset 3

Intermediate and final RPKM values for mouse muscle. (TXT 1423 kb)

Supplementary Dataset 4

Top 500 genes with strong multiread contributions in mouse liver. (XLS 52 kb)

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Mortazavi, A., Williams, B., McCue, K. et al. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5, 621–628 (2008). https://doi.org/10.1038/nmeth.1226

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