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Nature methods DOI:10.1038/nmeth.1491

Comprehensive comparative analysis of strand-specific RNA sequencing methods.

Publication TypeJournal Article
Year of Publication2010
AuthorsLevin, JZ, Yassour, M, Adiconis, X, Nusbaum, C, Thompson, DA, Friedman, N, Gnirke, A, Regev, A
JournalNature methods
Volume7
Issue9
Pages709-15
Date Published2010/09/01
ISSN1548-7091
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.

URLhttp://dx.doi.org/10.1038/nmeth.1491
DOI10.1038/nmeth.1491
Pubmed

http://www.ncbi.nlm.nih.gov/pubmed/20711195?dopt=Abstract