Comprehensive comparative analysis of strand-specific RNA sequencing methods


Strand-specific massively-parallel cDNA sequencing (RNA-Seq) is a powerful tool for novel transcript discovery, genome annotation, and expression profiling.  Despite multiple published methods for strand-specific RNA-Seq, no consensus exists as to how to choose between them.  Here, we developed a comprehensive computational pipeline for the comparison of 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 novel methods.  We found marked differences in complexity, strand-specificity, evenness and continuity of coverage, agreement with known annotations, and accuracy for expression profiling. Weighing each method’s performance and ease, we identify the dUTP second strand marking and the Illumina RNA ligation methods as the leading protocols, with the former benefitting from the availability of paired-end sequencing.  Our analysis provides a comprehensive benchmark, and our computational pipeline is applicable for assessment of future protocols in any organism.


Joshua Z. Levin1,*, Moran Yassour1,2,3,*, Xian Adiconis1, Chad Nusbaum1, Dawn Anne Thompson1, Nir Friedman3,4, Andreas Gnirke1, and Aviv Regev1,2,5

1 Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA  02142 USA

2 Department of Biology, Massachusetts Institute of Technology

3 School of Engineering and Computer Science, Hebrew University, Jerusalem, Israel

4 Alexander Silberman Institute of Life Sciences, Hebrew University, Jerusalem, Israel

5 Howard Hughes Medical Institute

*  These authors contributed equally to this work

Correspondence should be addressed to J.Z.L. ( and A.R. (