期刊
NATURE METHODS
卷 10, 期 7, 页码 623-+出版社
NATURE PORTFOLIO
DOI: 10.1038/nmeth.2483
关键词
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资金
- US National Institutes of Health [DP1-OD003958-01]
- US National Human Genome Research Institute (NHGRI) [1P01HG005062-01]
- NHGRI Center of Excellence in Genome Science [1P50HG006193-01]
- Howard Hughes Medical Institute
- Merkin Foundation for Stem Cell Research
- Klarman Cell Observatory at the Broad Institute
- NHGRI [HG03067]
- Fund for Scientific Research-Flanders (FWO Vlaanderen)
RNA-seq is an effective method for studying the transcriptome, but it 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 the relative merits of these methods 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 compared them against two control libraries. We found that the RNase H method performed best for chemically fragmented, low-quality RNA, and we confirmed this through analysis of actual degraded samples. RNase H can even effectively replace oligo(dT)-based methods for standard RNA-seq. SMART and NuGEN had distinct strengths for measuring low-quantity RNA. Our analysis allows biologists to select the most suitable methods and provides a benchmark for future method development.
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