Comprehensive comparative analysis of strand-specific RNA sequencing methods

Joshua Z. Levin, Moran Yassour, Xian Adiconis, Chad Nusbaum, Dawn Anne Thompson, Nir Friedman, Andreas Gnirke, Aviv Regev

Research output: Contribution to journalArticlepeer-review

569 Scopus citations


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.

Original languageAmerican English
Pages (from-to)709-715
Number of pages7
JournalNature Methods
Issue number9
StatePublished - Sep 2010

Bibliographical note

Funding Information:
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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