title: Evaluating methods for isolating total RNA and predicting the success of sequencing phylogenetically diverse plant transcriptomes.
abstract: Next-generation sequencing plays a central role in the characterization and quantification of transcriptomes. Although numerous metrics are purported to quantify the quality of RNA, there have been no large-scale empirical evaluations of the major determinants of sequencing success. We used a combination of existing and newly developed methods to isolate total RNA from 1115 samples from 695 plant species in 324 families, which represents >900 million years of phylogenetic diversity from green algae through flowering plants, including many plants of economic importance. We then sequenced 629 of these samples on Illumina GAIIx and HiSeq platforms and performed a large comparative analysis to identify predictors of RNA quality and the diversity of putative genes (scaffolds) expressed within samples. Tissue types (e.g., leaf vs. flower) varied in RNA quality, sequencing depth and the number of scaffolds. Tissue age also influenced RNA quality but not the number of scaffolds ≥ 1000 bp. Overall, 36% of the variation in the number of scaffolds was explained by metrics of RNA integrity (RIN score), RNA purity (OD 260/230), sequencing platform (GAIIx vs HiSeq) and the amount of total RNA used for sequencing. However, our results show that the most commonly used measures of RNA quality (e.g., RIN) are weak predictors of the number of scaffolds because Illumina sequencing is robust to variation in RNA quality. These results provide novel insight into the methods that are most important in isolating high quality RNA for sequencing and assembling plant transcriptomes. The methods and recommendations provided here could increase the efficiency and decrease the cost of RNA sequencing for individual labs and genome centers.
authors.affiliation: Department of Biology, University of Toronto at Mississauga, Mississauga, Ontario, Canada.
authors.forename: Marc T J
authors.initials: MT
authors.lastname: Johnson
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Carpenter
authors.forename: Zhijian
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authors.lastname: Tian
authors.forename: Richard
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authors.lastname: Bruskiewich
authors.forename: Jason N
authors.initials: JN
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authors.forename: Charlotte T
authors.initials: CT
authors.lastname: Carrigan
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authors.forename: Neil D
authors.initials: ND
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authors.forename: Sarah
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authors.initials: CW
authors.lastname: Depamphilis
authors.forename: Patrick P
authors.initials: PP
authors.lastname: Edger
authors.forename: Falicia
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authors.lastname: Goh
authors.forename: Sean
authors.initials: S
authors.lastname: Graham
authors.forename: Stephan
authors.initials: S
authors.lastname: Greiner
authors.forename: Julian M
authors.initials: JM
authors.lastname: Hibberd
authors.forename: Ingrid
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authors.lastname: Jordon-Thaden
authors.forename: Toni M
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authors.lastname: Kutchan
authors.forename: James
authors.initials: J
authors.lastname: Leebens-Mack
authors.forename: Michael
authors.initials: M
authors.lastname: Melkonian
authors.forename: Nicholas
authors.initials: N
authors.lastname: Miles
authors.forename: Henrietta
authors.initials: H
authors.lastname: Myburg
authors.forename: Jordan
authors.initials: J
authors.lastname: Patterson
authors.forename: J Chris
authors.initials: JC
authors.lastname: Pires
authors.forename: Paula
authors.initials: P
authors.lastname: Ralph
authors.forename: Megan
authors.initials: M
authors.lastname: Rolf
authors.forename: Rowan F
authors.initials: RF
authors.lastname: Sage
authors.forename: Douglas
authors.initials: D
authors.lastname: Soltis
authors.forename: Pamela
authors.initials: P
authors.lastname: Soltis
authors.forename: Dennis
authors.initials: D
authors.lastname: Stevenson
authors.forename: C Neal
authors.initials: CN
authors.lastname: Stewart
authors.forename: Barbara
authors.initials: B
authors.lastname: Surek
authors.forename: Christina J M
authors.initials: CJ
authors.lastname: Thomsen
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Villarreal
authors.forename: Xiaolei
authors.initials: X
authors.lastname: Wu
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
authors.forename: Michael K
authors.initials: MK
authors.lastname: Deyholos
authors.forename: Gane Ka-Shu
authors.initials: GK
authors.lastname: Wong
mesh_terms: D001483:Base Sequence; D035264:Flowers / Q000235:genetics*; D020869:Gene Expression Profiling; D018745:Genome, Plant; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods / Q000592:standards*; D010802:Phylogeny; D018515:Plant Leaves / Q000235:genetics*; D010944:Plants / Q000145:classification / Q000235:genetics*; D018749:RNA, Plant / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification* / Q000592:standards; D017423:Sequence Analysis, RNA
chemical_list: D018749:RNA, Plant