Johnson, Marc T. J., Carpenter, Eric J., Tian, Zhijian, Bruskiewich, Richard, Burris, Jason N., Carrigan, Charlotte T., Chase, Mark W., Clarke, Neil D., Covshoff, Sarah, dePamphilis, Claude W., Edger, Patrick P., Goh, Falicia ORCID: 0000-0002-8373-7408, Graham, Sean, Greiner, Stephan ORCID: 0000-0003-3369-0354, Hibberd, Julian M., Jordon-Thaden, Ingrid ORCID: 0000-0001-6225-6034, Kutchan, Toni M., Leebens-Mack, James ORCID: 0000-0003-4811-2231, Melkonian, Michael, Miles, Nicholas, Myburg, Henrietta, Patterson, Jordan, Pires, J. Chris ORCID: 0000-0001-9682-2639, Ralph, Paula, Rolf, Megan, Sage, Rowan F., Soltis, Douglas, Soltis, Pamela, Stevenson, Dennis, Stewart, C. Neal, Jr., Surek, Barbara, Thomsen, Christina J. M., Villarreal, Juan Carlos, Wu, Xiaolei, Zhang, Yong, Deyholos, Michael K. and Wong, Gane Ka-Shu (2012). Evaluating Methods for Isolating Total RNA and Predicting the Success of Sequencing Phylogenetically Diverse Plant Transcriptomes. PLoS One, 7 (11). SAN FRANCISCO: PUBLIC LIBRARY SCIENCE. ISSN 1932-6203

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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.

Item Type: Journal Article
Creators:
CreatorsEmailORCIDORCID Put Code
Johnson, Marc T. J.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Carpenter, Eric J.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Tian, ZhijianUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Bruskiewich, RichardUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Burris, Jason N.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Carrigan, Charlotte T.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Chase, Mark W.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Clarke, Neil D.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Covshoff, SarahUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
dePamphilis, Claude W.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Edger, Patrick P.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Goh, FaliciaUNSPECIFIEDorcid.org/0000-0002-8373-7408UNSPECIFIED
Graham, SeanUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Greiner, StephanUNSPECIFIEDorcid.org/0000-0003-3369-0354UNSPECIFIED
Hibberd, Julian M.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Jordon-Thaden, IngridUNSPECIFIEDorcid.org/0000-0001-6225-6034UNSPECIFIED
Kutchan, Toni M.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Leebens-Mack, JamesUNSPECIFIEDorcid.org/0000-0003-4811-2231UNSPECIFIED
Melkonian, MichaelUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Miles, NicholasUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Myburg, HenriettaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Patterson, JordanUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Pires, J. ChrisUNSPECIFIEDorcid.org/0000-0001-9682-2639UNSPECIFIED
Ralph, PaulaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Rolf, MeganUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Sage, Rowan F.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Soltis, DouglasUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Soltis, PamelaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Stevenson, DennisUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Stewart, C. Neal, Jr.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Surek, BarbaraUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Thomsen, Christina J. M.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Villarreal, Juan CarlosUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Wu, XiaoleiUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Zhang, YongUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Deyholos, Michael K.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Wong, Gane Ka-ShuUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
URN: urn:nbn:de:hbz:38-478502
DOI: 10.1371/journal.pone.0050226
Journal or Publication Title: PLoS One
Volume: 7
Number: 11
Date: 2012
Publisher: PUBLIC LIBRARY SCIENCE
Place of Publication: SAN FRANCISCO
ISSN: 1932-6203
Language: English
Faculty: Unspecified
Divisions: Unspecified
Subjects: no entry
Uncontrolled Keywords:
KeywordsLanguage
REVEALS; ECOLOGY; SEQ; MAPMultiple languages
Multidisciplinary SciencesMultiple languages
URI: http://kups.ub.uni-koeln.de/id/eprint/47850

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