Email updates

Keep up to date with the latest news and content from BMC Proceedings and BioMed Central.

This article is part of the supplement: Beyond the Genome 2012

Open Access Open Badges Poster presentation

Comparative analysis of strand-specific RNA sequencing approaches

Daniela Munafo*, Ping Liu, Christine Sumner, Erbay Yigit, Landon Merrill, Lynne Apone, Brad Langhorst, Fiona Stewart, Eileen T Dimalanta and Theodore Davis

  • * Corresponding author: Daniela Munafo

Author Affiliations

New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA

For all author emails, please log on.

BMC Proceedings 2012, 6(Suppl 6):P30  doi:10.1186/1753-6561-6-S6-P30

The electronic version of this article is the complete one and can be found online at:

Published:1 October 2012

© 2012 Munafo et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Standard RNA sequencing approaches generally require double-stranded cDNA synthesis, which erases RNA strand information.

Synthesis of a randomly primed double-stranded cDNA followed by addition of adaptors for next-generation sequencing leads to the loss of information about which strand was present in the original mRNA template. The polarity of the transcript is important for correct annotation of novel genes, identification of antisense transcripts with potential regulatory roles, and for correct determination of gene expression levels in the presence of antisense transcripts. Different strand-specific RNA-seq approaches have been developed to preserve information about strand polarity with different level of performances.

Material and methods

Using Illumina Deep Sequencing Technology, this work investigates the performance of two different directional RNA-Seq (strand-specific RNA-seq) strategies. One is based on direct ligation of adaptors to the RNA ends and the other is based on the labeling and excision of the second strand cDNA. The RNA-seq workflows present in this work have been improved over current more laborious RNA-seq methods. Their low RNA input and streamlined workflows make them compatible with high throughput and automation. We also analyze the effect of different RNA fragmentation methods (divalent cations plus heat versus enzymatic fragmentation).


We will provide a comparative full data analysis of different strand-specific RNA methods (library performance, complexity, continuity of gene coverage, strand specificity, rRNA background).


Our results show improved methods for high-quality strand-specific RNA-seq library construction amenable to large-scale library construction and automation.