Evaluating methods for isolating total rna and predicting the success of sequencing phylogenetically diverse plant transcriptomes
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 combin...
| Autores principales: | , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , |
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| Formato: | Journal Article |
| Lenguaje: | Inglés |
| Publicado: |
Public Library of Science
2012
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| Materias: | |
| Acceso en línea: | https://hdl.handle.net/10568/165718 |
| _version_ | 1855524651264901120 |
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| author | 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 Graham, Sean Greiner, Stephan Hibberd, Julian M. Jordon-Thaden, Ingrid Kutchan, Toni M. Leebens-Mack, James Melkonian, Michael Miles, Nicholas Myburg, Henrietta Patterson, Jordan Pires, J. Chris Ralph, Paula Rolf, Megan Sage, Rowan F. Soltis, Douglas Soltis, Pamela Stevenson, Dennis Stewart, C. Neal Surek, Barbara Thomsen, Christina J. M. Villarreal, Juan Carlos Wu, Xiaolei Zhang, Yong Deyholos, Michael K. Wong, Gane Ka-Shu |
| author_browse | Bruskiewich, Richard Burris, Jason N. Carpenter, Eric J. Carrigan, Charlotte T. Chase, Mark W. Clarke, Neil D. Covshoff, Sarah Deyholos, Michael K. Edger, Patrick P. Goh, Falicia Graham, Sean Greiner, Stephan Hibberd, Julian M. Johnson, Marc T. J. Jordon-Thaden, Ingrid Kutchan, Toni M. Leebens-Mack, James Melkonian, Michael Miles, Nicholas Myburg, Henrietta Patterson, Jordan Pires, J. Chris Ralph, Paula Rolf, Megan Sage, Rowan F. Soltis, Douglas Soltis, Pamela Stevenson, Dennis Stewart, C. Neal Surek, Barbara Thomsen, Christina J. M. Tian, Zhijian Villarreal, Juan Carlos Wong, Gane Ka-Shu Wu, Xiaolei Zhang, Yong dePamphilis, Claude W. |
| author_facet | 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 Graham, Sean Greiner, Stephan Hibberd, Julian M. Jordon-Thaden, Ingrid Kutchan, Toni M. Leebens-Mack, James Melkonian, Michael Miles, Nicholas Myburg, Henrietta Patterson, Jordan Pires, J. Chris Ralph, Paula Rolf, Megan Sage, Rowan F. Soltis, Douglas Soltis, Pamela Stevenson, Dennis Stewart, C. Neal Surek, Barbara Thomsen, Christina J. M. Villarreal, Juan Carlos Wu, Xiaolei Zhang, Yong Deyholos, Michael K. Wong, Gane Ka-Shu |
| author_sort | Johnson, Marc T. J. |
| collection | Repository of Agricultural Research Outputs (CGSpace) |
| description | 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. |
| format | Journal Article |
| id | CGSpace165718 |
| institution | CGIAR Consortium |
| language | Inglés |
| publishDate | 2012 |
| publishDateRange | 2012 |
| publishDateSort | 2012 |
| publisher | Public Library of Science |
| publisherStr | Public Library of Science |
| record_format | dspace |
| spelling | CGSpace1657182025-01-24T14:12:51Z Evaluating methods for isolating total rna and predicting the success of sequencing phylogenetically diverse plant transcriptomes 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 Graham, Sean Greiner, Stephan Hibberd, Julian M. Jordon-Thaden, Ingrid Kutchan, Toni M. Leebens-Mack, James Melkonian, Michael Miles, Nicholas Myburg, Henrietta Patterson, Jordan Pires, J. Chris Ralph, Paula Rolf, Megan Sage, Rowan F. Soltis, Douglas Soltis, Pamela Stevenson, Dennis Stewart, C. Neal Surek, Barbara Thomsen, Christina J. M. Villarreal, Juan Carlos Wu, Xiaolei Zhang, Yong Deyholos, Michael K. Wong, Gane Ka-Shu gene expression genes genetic variation nucleotide sequences phylogeny rna transcriptomes transcriptomics 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. 2012-11-21 2024-12-19T12:55:23Z 2024-12-19T12:55:23Z Journal Article https://hdl.handle.net/10568/165718 en Open Access Public Library of Science 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; Graham, Sean; Greiner, Stephan; Hibberd, Julian M.; Jordon-Thaden, Ingrid; Kutchan, Toni M.; Leebens-Mack, James; Melkonian, Michael; Miles, Nicholas; Myburg, Henrietta; Patterson, Jordan; Pires, J. Chris; Ralph, Paula; Rolf, Megan; Sage, Rowan F.; Soltis, Douglas; Soltis, Pamela; Stevenson, Dennis; Stewart, C. Neal; 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, Volume 7 no. 11 p. e50226 |
| spellingShingle | gene expression genes genetic variation nucleotide sequences phylogeny rna transcriptomes transcriptomics 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 Graham, Sean Greiner, Stephan Hibberd, Julian M. Jordon-Thaden, Ingrid Kutchan, Toni M. Leebens-Mack, James Melkonian, Michael Miles, Nicholas Myburg, Henrietta Patterson, Jordan Pires, J. Chris Ralph, Paula Rolf, Megan Sage, Rowan F. Soltis, Douglas Soltis, Pamela Stevenson, Dennis Stewart, C. Neal Surek, Barbara Thomsen, Christina J. M. Villarreal, Juan Carlos Wu, Xiaolei Zhang, Yong Deyholos, Michael K. Wong, Gane Ka-Shu Evaluating methods for isolating total rna and predicting the success of sequencing phylogenetically diverse plant transcriptomes |
| title | Evaluating methods for isolating total rna and predicting the success of sequencing phylogenetically diverse plant transcriptomes |
| title_full | Evaluating methods for isolating total rna and predicting the success of sequencing phylogenetically diverse plant transcriptomes |
| title_fullStr | Evaluating methods for isolating total rna and predicting the success of sequencing phylogenetically diverse plant transcriptomes |
| title_full_unstemmed | Evaluating methods for isolating total rna and predicting the success of sequencing phylogenetically diverse plant transcriptomes |
| title_short | Evaluating methods for isolating total rna and predicting the success of sequencing phylogenetically diverse plant transcriptomes |
| title_sort | evaluating methods for isolating total rna and predicting the success of sequencing phylogenetically diverse plant transcriptomes |
| topic | gene expression genes genetic variation nucleotide sequences phylogeny rna transcriptomes transcriptomics |
| url | https://hdl.handle.net/10568/165718 |
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