title: Comparative analysis of RNA sequencing methods for degraded or low-input samples.
abstract: 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.
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
authors.forename: Xian
authors.initials: X
authors.lastname: Adiconis
authors.forename: Diego
authors.initials: D
authors.lastname: Borges-Rivera
authors.forename: Rahul
authors.initials: R
authors.lastname: Satija
authors.forename: David S
authors.initials: DS
authors.lastname: DeLuca
authors.forename: Michele A
authors.initials: MA
authors.lastname: Busby
authors.forename: Aaron M
authors.initials: AM
authors.lastname: Berlin
authors.forename: Andrey
authors.initials: A
authors.lastname: Sivachenko
authors.forename: Dawn Anne
authors.initials: DA
authors.lastname: Thompson
authors.forename: Alec
authors.initials: A
authors.lastname: Wysoker
authors.forename: Timothy
authors.initials: T
authors.lastname: Fennell
authors.forename: Andreas
authors.initials: A
authors.lastname: Gnirke
authors.forename: Nathalie
authors.initials: N
authors.lastname: Pochet
authors.forename: Aviv
authors.initials: A
authors.lastname: Regev
authors.forename: Joshua Z
authors.initials: JZ
authors.lastname: Levin
mesh_terms: D000465:Algorithms; D016477:Artifacts; D020869:Gene Expression Profiling / Q000379:methods*; D012313:RNA / Q000235:genetics*; D018401:Sample Size; D017423:Sequence Analysis, RNA / Q000379:methods*; D012984:Software; D059467:Transcriptome / Q000235:genetics*
chemical_list: D012313:RNA