Comprehensive comparative analysis of RNA sequencing methods for degraded or low input samples
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Author
Adiconis, Xian
Borges-Rivera, Diego
Satija, Rahul
DeLuca, David S.
Busby, Michele A.
Berlin, Aaron M.
Sivachenko, Andrey
Thompson, Dawn Anne
Wysoker, Alec
Fennell, Timothy
Gnirke, Andreas
Pochet, Nathalie
Regev, Aviv
Note: Order does not necessarily reflect citation order of authors.
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https://doi.org/10.1038/nmeth.2483Metadata
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Adiconis, X., D. Borges-Rivera, R. Satija, D. S. DeLuca, M. A. Busby, A. M. Berlin, A. Sivachenko, et al. 2013. “Comprehensive comparative analysis of RNA sequencing methods for degraded or low input samples.” Nature methods 10 (7): 10.1038/nmeth.2483. doi:10.1038/nmeth.2483. http://dx.doi.org/10.1038/nmeth.2483.Abstract
RNA-Seq is an effective method to study the transcriptome, but can be difficult to apply to scarce or degraded RNA from fixed clinical samples, rare cell populations, or cadavers. Recent studies have proposed several methods for RNA-Seq of low quality and/or low quantity samples, but their relative merits have not been systematically analyzed. Here, we compare five such methods using metrics relevant to transcriptome annotation, transcript discovery, and gene expression. Using a single human RNA sample, we constructed and sequenced ten libraries with these methods and two control libraries. We find that the RNase H method performed best for low quality RNA, and confirmed this with actual degraded samples. RNase H can even effectively replace oligo (dT) based methods for standard RNA-Seq. SMART and NuGEN had distinct strengths for low quantity RNA. Our analysis allows biologists to select the most suitable methods and provides a benchmark for future method development.Other Sources
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3821180/pdf/Terms of Use
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