Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Analysis
  • Published:

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

Accession codes


Gene Expression Omnibus


  1. Wang, Z., Gerstein, M. & Snyder, M. RNA-Seq: a revolutionary tool for transcriptomics. Nat. Rev. Genet. 10, 57–63 (2009).

    Article  CAS  Google Scholar 

  2. Wilhelm, B.T. et al. Dynamic repertoire of a eukaryotic transcriptome surveyed at single-nucleotide resolution. Nature 453, 1239–1243 (2008).

    Article  CAS  Google Scholar 

  3. Denoeud, F. et al. Annotating genomes with massive-scale RNA sequencing. Genome Biol. 9, R175 (2008).

    Article  Google Scholar 

  4. Yassour, M. et al. Ab initio construction of a eukaryotic transcriptome by massively parallel mRNA sequencing. Proc. Natl. Acad. Sci. USA 106, 3264–3269 (2009).

    Article  CAS  Google Scholar 

  5. Marioni, J.C., Mason, C.E., Mane, S.M., Stephens, M. & Gilad, Y. RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays. Genome Res. 18, 1509–1517 (2008).

    Article  CAS  Google Scholar 

  6. Mortazavi, A., Williams, B.A., McCue, K., Schaeffer, L. & Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods 5, 621–628 (2008).

    Article  CAS  Google Scholar 

  7. Pan, Q., Shai, O., Lee, L.J., Frey, B.J. & Blencowe, B.J. Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat. Genet. 40, 1413–1415 (2008).

    Article  CAS  Google Scholar 

  8. Wang, E.T. et al. Alternative isoform regulation in human tissue transcriptomes. Nature 456, 470–476 (2008).

    Article  CAS  Google Scholar 

  9. Sultan, M. et al. A global view of gene activity and alternative splicing by deep sequencing of the human transcriptome. Science 321, 956–960 (2008).

    Article  CAS  Google Scholar 

  10. Guttman, M. et al. Ab initio reconstruction of cell type-specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAs. Nat. Biotechnol. 28, 503–510 (2010).

    Article  CAS  Google Scholar 

  11. Trapnell, C. et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010).

    Article  CAS  Google Scholar 

  12. Core, L.J., Waterfall, J.J. & Lis, J.T. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322, 1845–1848 (2008).

    Article  CAS  Google Scholar 

  13. Parkhomchuk, D. et al. Transcriptome analysis by strand-specific sequencing of complementary DNA. Nucleic Acids Res. 37, e123 (2009).

    Article  Google Scholar 

  14. Ingolia, N.T., Ghaemmaghami, S., Newman, J.R. & Weissman, J.S. Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324, 218–223 (2009).

    Article  CAS  Google Scholar 

  15. He, Y., Vogelstein, B., Velculescu, V.E., Papadopoulos, N. & Kinzler, K.W. The antisense transcriptomes of human cells. Science 322, 1855–1857 (2008).

    Article  CAS  Google Scholar 

  16. Schaefer, M., Pollex, T., Hanna, K. & Lyko, F. RNA cytosine methylation analysis by bisulfite sequencing. Nucleic Acids Res. 37, e12 (2009).

    Article  Google Scholar 

  17. Jaffe, D.B. et al. Whole-genome sequence assembly for mammalian genomes: Arachne 2. Genome Res. 13, 91–96 (2003).

    Article  CAS  Google Scholar 

  18. Xu, Z. et al. Bidirectional promoters generate pervasive transcription in yeast. Nature 457, 1033–1037 (2009).

    Article  CAS  Google Scholar 

  19. Guo, J., Wu, T., Bess, J., Henderson, L.E. & Levin, J.G. Actinomycin D inhibits human immunodeficiency virus type 1 minus-strand transfer in in vitro and endogenous reverse transcriptase assays. J. Virol. 72, 6716–6724 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Gentleman, R., Carey, V., Huber, W., Irizarry, R. & Dudoit, S. (eds.). Bioinformatics and Computational Biology Solutions Using R and Bioconductor, 473 (Springer, Secaucus, NJ, 2005).

  21. Yang, Y.H. et al. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res. 30, e15 (2002).

    Article  Google Scholar 

  22. Croucher, N.J. et al. A simple method for directional transcriptome sequencing using Illumina technology. Nucleic Acids Res. 37, e148 (2009).

    Article  Google Scholar 

  23. Lipson, D. et al. Quantification of the yeast transcriptome by single-molecule sequencing. Nat. Biotechnol. 27, 652–658 (2009).

    Article  CAS  Google Scholar 

  24. Ozsolak, F. et al. Direct RNA sequencing. Nature 461, 814–818 (2009).

    Article  CAS  Google Scholar 

  25. Affymetrix / Cold Spring Harbor Laboratory ENCODE Transcriptome Project. Post-transcriptional processing generates a diversity of 5′-modified long and short RNAs. Nature 457, 1028–1032 (2009).

  26. Li, H. et al. Determination of tag density required for digital transcriptome analysis: application to an androgen-sensitive prostate cancer model. Proc. Natl. Acad. Sci. USA 105, 20179–20184 (2008).

    Article  CAS  Google Scholar 

  27. Mamanova, L. et al. FRT-seq: amplification-free, strand-specific transcriptome sequencing. Nat. Methods 7, 130–132 (2010).

    Article  CAS  Google Scholar 

  28. Linsen, S.E. et al. Limitations and possibilities of small RNA digital gene expression profiling. Nat. Methods 6, 474–476 (2009).

    Article  CAS  Google Scholar 

  29. Lister, R. et al. Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell 133, 523–536 (2008).

    Article  CAS  Google Scholar 

  30. Zhu, Y.Y., Machleder, E.M., Chenchik, A., Li, R. & Siebert, P.D. Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction. Biotechniques 30, 892–897 (2001).

    Article  CAS  Google Scholar 

  31. Armour, C.D. et al. Digital transcriptome profiling using selective hexamer priming for cDNA synthesis. Nat. Methods 6, 647–649 (2009).

    Article  CAS  Google Scholar 

  32. Cloonan, N. et al. Stem cell transcriptome profiling via massive-scale mRNA sequencing. Nat. Methods 5, 613–619 (2008).

    Article  CAS  Google Scholar 

Download references


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.).

Author information

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.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

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

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing