JohnsonCarpenterTianEtAl2012

Référence

Johnson, M.T. J., Carpenter, E.J., Tian, Z., Bruskiewich, R., Burris, J.N., Carrigan, C.T., Chase, M.W., Clarke, N.D., Covshoff, S., dePamphilis, C.W., Edger, P.P., Goh, F., Graham, S., Greiner, S., Hibberd, J.M., Jordon-Thaden, I., Kutchan, T.M., Leebens-Mack, J., Melkonian, M., Miles, N., Myburg, H., Patterson, J., Pires, J. C., Ralph, P., Rolf, M., Sage, F., Soltis, D., Soltis, P., Stevenson, D., Stewart, Jr., C.N., Surek, B., Thomsen, C. J. M., Villarreal, J.C., Wu, X., Zhang, Y., Deyholos, M.K. and Wong, G.K.-S. (2012) Evaluating Methods for Isolating Total RNA and Predicting the Success of Sequencing Phylogenetically Diverse Plant Transcriptomes. PLoS ONE, 7(11).

Résumé

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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@ARTICLE { JohnsonCarpenterTianEtAl2012,
    AUTHOR = { Johnson, M.T. J. and Carpenter, E.J. and Tian, Z. and Bruskiewich, R. and Burris, J.N. and Carrigan, C.T. and Chase, M.W. and Clarke, N.D. and Covshoff, S. and dePamphilis, C.W. and Edger, P.P. and Goh, F. and Graham, S. and Greiner, S. and Hibberd, J.M. and Jordon-Thaden, I. and Kutchan, T.M. and Leebens-Mack, J. and Melkonian, M. and Miles, N. and Myburg, H. and Patterson, J. and Pires, J. C. and Ralph, P. and Rolf, M. and Sage, F. and Soltis, D. and Soltis, P. and Stevenson, D. and Stewart, Jr., C.N. and Surek, B. and Thomsen, C. J. M. and Villarreal, J.C. and Wu, X. and Zhang, Y. and Deyholos, M.K. and Wong, G.K.-S. },
    TITLE = { Evaluating Methods for Isolating Total RNA and Predicting the Success of Sequencing Phylogenetically Diverse Plant Transcriptomes },
    JOURNAL = { PLoS ONE },
    YEAR = { 2012 },
    VOLUME = { 7 },
    NUMBER = { 11 },
    MONTH = { nov },
    ISSN = { 1932-6203 },
    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. },
    ARTICLE-NUMBER = { e50226 },
    DOI = { 10.1371/journal.pone.0050226 },
    ORCID-NUMBERS = { Wong, Gane Ka-Shu/0000-0001-6108-5560 Villarreal A., Juan Carlos/0000-0002-0770-1446 Graham, Sean/0000-0001-8209-5231 Pires, J Chris/0000-0001-9682-2639 },
    RESEARCHERID-NUMBERS = { Wong, Gane Ka-Shu/G-5784-2013 Villarreal A., Juan Carlos/C-6277-2018 Kutchan, Toni/L-4625-2013 Jordon-Thaden, Ingrid/A-5619-2012 Chase, Mark/A-6642-2011 Graham, Sean/L-3944-2014 Clarke, Neil/D-9613-2014 dePamphilis, Claude/P-6652-2016 },
    UNIQUE-ID = { ISI:000311821000204 },
}

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