Comprehensive comparative analysis of strand-specific RNA sequencing methods
Strand-specific, massively parallel cDNA sequencing (RNA-seq) is a powerful tool for transcript discovery, genome annotation and expression profiling. There are multiple published methods for strand-specific RNA-seq, but no consensus exists as to how to choose between them. Here we developed a compr...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | en_US |
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Nature Publishing Group
2012
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| Online Access: | http://hdl.handle.net/1721.1/74517 https://orcid.org/0000-0001-8567-2049 |
| _version_ | 1826200049146658816 |
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| author | Regev, Aviv Levin, Joshua Z. Yassour, Moran Adiconis, Xian Nusbaum, Chad Thompson, Dawn Anne Friedman, Nir Gnirke, Andreas |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Regev, Aviv Levin, Joshua Z. Yassour, Moran Adiconis, Xian Nusbaum, Chad Thompson, Dawn Anne Friedman, Nir Gnirke, Andreas |
| author_sort | Regev, Aviv |
| collection | MIT |
| description | Strand-specific, massively parallel cDNA sequencing (RNA-seq) is a powerful tool for transcript discovery, genome annotation and expression profiling. There are multiple published methods for strand-specific RNA-seq, but no consensus exists as to how to choose between them. Here we developed a comprehensive computational pipeline to compare library quality metrics from any RNA-seq method. Using the well-annotated Saccharomyces cerevisiae transcriptome as a benchmark, we compared seven library-construction protocols, including both published and our own methods. We found marked differences in strand specificity, library complexity, evenness and continuity of coverage, agreement with known annotations and accuracy for expression profiling. Weighing each method's performance and ease, we identified the dUTP second-strand marking and the Illumina RNA ligation methods as the leading protocols, with the former benefitting from the current availability of paired-end sequencing. Our analysis provides a comprehensive benchmark, and our computational pipeline is applicable for assessment of future protocols in other organisms. |
| first_indexed | 2024-09-23T11:30:08Z |
| format | Article |
| id | mit-1721.1/74517 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T11:30:08Z |
| publishDate | 2012 |
| publisher | Nature Publishing Group |
| record_format | dspace |
| spelling | mit-1721.1/745172022-10-01T04:01:42Z Comprehensive comparative analysis of strand-specific RNA sequencing methods Regev, Aviv Levin, Joshua Z. Yassour, Moran Adiconis, Xian Nusbaum, Chad Thompson, Dawn Anne Friedman, Nir Gnirke, Andreas Massachusetts Institute of Technology. Department of Biology Regev, Aviv Strand-specific, massively parallel cDNA sequencing (RNA-seq) is a powerful tool for transcript discovery, genome annotation and expression profiling. There are multiple published methods for strand-specific RNA-seq, but no consensus exists as to how to choose between them. Here we developed a comprehensive computational pipeline to compare library quality metrics from any RNA-seq method. Using the well-annotated Saccharomyces cerevisiae transcriptome as a benchmark, we compared seven library-construction protocols, including both published and our own methods. We found marked differences in strand specificity, library complexity, evenness and continuity of coverage, agreement with known annotations and accuracy for expression profiling. Weighing each method's performance and ease, we identified the dUTP second-strand marking and the Illumina RNA ligation methods as the leading protocols, with the former benefitting from the current availability of paired-end sequencing. Our analysis provides a comprehensive benchmark, and our computational pipeline is applicable for assessment of future protocols in other organisms. Howard Hughes Medical Institute United States-Israel Binational Science Foundation 2012-10-30T17:15:35Z 2012-10-30T17:15:35Z 2010-08 2010-03 Article http://purl.org/eprint/type/JournalArticle 1548-7091 1548-7105 http://hdl.handle.net/1721.1/74517 Levin, Joshua Z et al. “Comprehensive Comparative Analysis of Strand-specific RNA Sequencing Methods.” Nature Methods 7.9 (2010): 709–715. https://orcid.org/0000-0001-8567-2049 en_US http://dx.doi.org/10.1038/nmeth.1491 Nature Methods Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf Nature Publishing Group PMC |
| spellingShingle | Regev, Aviv Levin, Joshua Z. Yassour, Moran Adiconis, Xian Nusbaum, Chad Thompson, Dawn Anne Friedman, Nir Gnirke, Andreas Comprehensive comparative analysis of strand-specific RNA sequencing methods |
| title | Comprehensive comparative analysis of strand-specific RNA sequencing methods |
| title_full | Comprehensive comparative analysis of strand-specific RNA sequencing methods |
| title_fullStr | Comprehensive comparative analysis of strand-specific RNA sequencing methods |
| title_full_unstemmed | Comprehensive comparative analysis of strand-specific RNA sequencing methods |
| title_short | Comprehensive comparative analysis of strand-specific RNA sequencing methods |
| title_sort | comprehensive comparative analysis of strand specific rna sequencing methods |
| url | http://hdl.handle.net/1721.1/74517 https://orcid.org/0000-0001-8567-2049 |
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