Comparative analysis of RNA sequencing methods for degraded or low-input samples.
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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U54 HG003067 / HG / NHGRI NIH HHS / United States
DP1-OD003958-01 / OD / NIH HHS / United States
F32 HD075541 / HD / NICHD NIH HHS / United States
1P01HG005062-01 / HG / NHGRI NIH HHS / United States
HG03067 / HG / NHGRI NIH HHS / United States
Howard Hughes Medical Institute / United States
P50 HG006193 / HG / NHGRI NIH HHS / United States
1P50HG006193-01 / HG / NHGRI NIH HHS / United States
P01 HG005062 / HG / NHGRI NIH HHS / United States
DP1 OD003958 / OD / NIH HHS / United States