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Comprehensive comparative analysis of strand-specific RNA sequencing methods


Strand-specific, massively parallel cDNA sequencing (RNA-seq) is a powerful tool for transcript discovery, genome annotation and expression profiling. There are multiple published methods for strand-specific RNA-seq, but no consensus exists as to how to choose between them. Here we developed a comprehensive computational pipeline to compare library quality metrics from any RNA-seq method. Using the well-annotated Saccharomyces cerevisiae transcriptome as a benchmark, we compared seven library-construction protocols, including both published and our own methods. We found marked differences in strand specificity, library complexity, evenness and continuity of coverage, agreement with known annotations and accuracy for expression profiling. Weighing each method's performance and ease, we identified the dUTP second-strand marking and the Illumina RNA ligation methods as the leading protocols, with the former benefitting from the current availability of paired-end sequencing. Our analysis provides a comprehensive benchmark, and our computational pipeline is applicable for assessment of future protocols in other organisms.

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Figure 1: Methods for strand-specific RNA-seq.
Figure 2: Key criteria for evaluation of strand-specific RNA-seq libraries.
Figure 3: Complexity of single- and paired-end libraries.
Figure 4: Strand specificity and evenness of transcript coverage.
Figure 5: Continuity of transcript coverage.
Figure 6: Digital expression profiling using strand-specific RNA-seq.

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We thank members of the Broad Genome Sequencing Platform for sequencing work, J. Meldrim for advice on monotemplate sequencing issues, T. Fennell for help with read processing, S. Luo and G. Schroth (Illumina) for sharing their Illumina RNA ligation protocol, L. Gaffney for assistance with figure graphics, J. Weissman for discussions and T. Liefeld and M. Reich for assistance with the GenePattern module. This work was supported by a US National Institutes of Health Director's Pioneer award, a Career Award at the Scientific Interface from the Burroughs Wellcome Fund, the Human Frontiers Science Program, a Sloan Fellowship, the Merkin Foundation for Stem Cell Research at the Broad Institute, and Howard Hughes Medical Institute (A.R.), by the US-Israel Binational Science Foundation (N.F. and A.R.), by the Canadian friends of the Hebrew University (M.Y.) and by US National Human Genome Research Institute grant HG03067-05 (C.N.).

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Authors and Affiliations



J.Z.L., M.Y., X.A., D.A.T., N.F. and A.R. wrote the paper. J.Z.L., M.Y., X.A., C.N., D.A.T., N.F., A.G. and A.R. assisted in editing the paper. D.A.T. prepared the poly(A)+ RNA. J.Z.L. and X.A. prepared the cDNA libraries. M.Y., N.F. and A.R. developed and performed the computational analysis. J.Z.L., X.A., M.Y., N.F. and A.R. conceived the research.

Corresponding authors

Correspondence to Joshua Z Levin or Aviv Regev.

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The authors declare no competing financial interests.

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Supplementary Figures 1–5, Supplementary Tables 1–5, Supplementary Notes 1–3 (PDF 1841 kb)

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Levin, J., Yassour, M., Adiconis, X. et al. Comprehensive comparative analysis of strand-specific RNA sequencing methods. Nat Methods 7, 709–715 (2010).

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