Comprehensive comparative analysis of strand-specific RNA sequencing methods
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Regev_Comprehensive comparative.pdf
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2.55 MB
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Author(s)
Regev, Aviv
Levin, Joshua Z.
Yassour, Moran
Adiconis, Xian
Nusbaum, Chad
Thompson, Dawn Anne
Friedman, Nir
Gnirke, Andreas
Date Issued
August 2010
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
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.
MIT Department
Massachusetts Institute of Technology. Department of Biology
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Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.
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DOI of Published Version
https://doi.org/10.1038/nmeth.1491
