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  Evaluating methods for isolating total RNA and predicting the success of sequencing phylogenetically diverse plant transcriptomes

Johnson, M. T., Carpenter, E. J., Tian, Z., Bruskiewich, R., Burris, J. N., Carrigan, C. T., et al. (2012). Evaluating methods for isolating total RNA and predicting the success of sequencing phylogenetically diverse plant transcriptomes. PLoS One, 7(11), e50226. doi:10.1371/journal.pone.0050226.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0014-1F79-4 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0019-7F67-B
Genre: Journal Article

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 Creators:
Johnson, M. T.1, Author
Carpenter, E. J.1, Author
Tian, Z.1, Author
Bruskiewich, R.1, Author
Burris, J. N.1, Author
Carrigan, C. T.1, Author
Chase, M. W.1, Author
Clarke, N. D.1, Author
Covshoff, S.1, Author
Depamphilis, C. W.1, Author
Edger, P. P.1, Author
Goh, F.1, Author
Graham, S.1, Author
Greiner, S.2, Author              
Hibberd, J. M.1, Author
Jordon-Thaden, I.1, Author
Kutchan, T. M.1, Author
Leebens-Mack, J.1, Author
Melkonian, M.1, Author
Miles, N.1, Author
Myburg, H.1, AuthorPatterson, J.1, AuthorPires, J. C.1, AuthorRalph, P.1, AuthorRolf, M.1, AuthorSage, R. F.1, AuthorSoltis, D.1, AuthorSoltis, P.1, AuthorStevenson, D.1, AuthorStewart, C. N., Jr.1, AuthorSurek, B.1, AuthorThomsen, C. J.1, AuthorVillarreal, J. C.1, AuthorWu, X.1, AuthorZhang, Y.1, AuthorDeyholos, M. K.1, AuthorWong, G. K.1, Author more..
Affiliations:
1External Organizations, ou_persistent22              
2Cytoplasmic and Evolutionary Genetics, Department Bock, Max Planck Institute of Molecular Plant Physiology, Max Planck Society, ou_1753324              

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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.

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Language(s): eng - English
 Dates: 2012-11-282012
 Publication Status: Published in print
 Pages: -
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Title: PLoS One
Source Genre: Journal
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Publ. Info: San Francisco, CA : Public Library of Science
Pages: - Volume / Issue: 7 (11) Sequence Number: - Start / End Page: e50226 Identifier: ISSN: 1932-6203
CoNE: https://pure.mpg.de/cone/journals/resource/1000000000277850