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Comprehensive comparative analysis of strand-specific RNA sequencing methods

Author(s)
Regev, Aviv; Levin, Joshua Z.; Yassour, Moran; Adiconis, Xian; Nusbaum, Chad; Thompson, Dawn Anne; Friedman, Nir; Gnirke, Andreas; ... Show more Show less
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Abstract
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.
Date issued
2010-08
URI
http://hdl.handle.net/1721.1/74517
Department
Massachusetts Institute of Technology. Department of Biology
Journal
Nature Methods
Publisher
Nature Publishing Group
Citation
Levin, Joshua Z et al. “Comprehensive Comparative Analysis of Strand-specific RNA Sequencing Methods.” Nature Methods 7.9 (2010): 709–715.
Version: Author's final manuscript
ISSN
1548-7091
1548-7105

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