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Modeling of RNA-seq fragment sequence bias reduces systematic errors in transcript abundance estimation

Abstract

We find that current computational methods for estimating transcript abundance from RNA-seq data can lead to hundreds of false-positive results. We show that these systematic errors stem largely from a failure to model fragment GC content bias. Sample-specific biases associated with fragment sequence features lead to misidentification of transcript isoforms. We introduce alpine, a method for estimating sample-specific bias-corrected transcript abundance. By incorporating fragment sequence features, alpine greatly increases the accuracy of transcript abundance estimates, enabling a fourfold reduction in the number of false positives for reported changes in expression compared with Cufflinks. Using simulated data, we also show that alpine retains the ability to discover true positives, similar to other approaches. The method is available as an R/Bioconductor package that includes data visualization tools useful for bias discovery.

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Figure 1: Quantification of transcript abundance from RNA-seq experiments.
Figure 2: Problems with current transcript abundance estimation methods.
Figure 3: Modeling and correcting fragment sequence bias.

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Acknowledgements

The authors are grateful for helpful suggestions from Y. Benjamini, W. Huber, N. Lahens, L. Pinello, C. Meyer, R. Patro, Z. Xu, and Y. Li. M.I.L. was supported by NIH grant 5T32CA009337-35. J.B.H. was supported by NIH R01 grant HG005220, the National Institute of Neurological Disorders and Stroke (5R01NS054794-08 to J.B.H.), the Defense Advanced Research Projects Agency (DARPA-D12AP00025, to John Harer, Duke University). R.A.I. was supported by NIH R01 grant HG005220.

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M.I.L. and R.A.I. designed the method. M.I.L., J.B.H., and R.A.I. wrote the manuscript.

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Correspondence to Rafael A Irizarry.

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The authors declare no competing financial interests.

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Supplementary Tables 1–5 and Supplementary Figures 1–25 (PDF 6827 kb)

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Love, M., Hogenesch, J. & Irizarry, R. Modeling of RNA-seq fragment sequence bias reduces systematic errors in transcript abundance estimation. Nat Biotechnol 34, 1287–1291 (2016). https://doi.org/10.1038/nbt.3682

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