Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction
Background Ribosomal RNA (rRNA) comprises at least 90% of total RNA extracted from mammalian tissue or cell line samples. Informative transcriptional profiling using massively parallel sequencing technologies requires either enrichment of mature poly-adenylated transcripts or target...
| Main Authors: | , , , , , , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
BioMed Central Ltd
2018
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| Online Access: | http://hdl.handle.net/1721.1/114726 |
| _version_ | 1811082767268577280 |
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| author | Herbert, Zachary T Kershner, Jamie P Thimmapuram, Jyothi Choudhari, Sulbha Alekseyev, Yuriy O Fan, Jun Podnar, Jessica W Wilcox, Edward Gipson, Jenny Gillaspy, Allison Jepsen, Kristen BonDurant, Sandra S Morris, Krystalynne Berkeley, Maura LeClerc, Ashley Simpson, Stephen D Sommerville, Gary Grimmett, Leslie Adams, Marie Butty, Vincent L G Levine, Stuart S. |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Herbert, Zachary T Kershner, Jamie P Thimmapuram, Jyothi Choudhari, Sulbha Alekseyev, Yuriy O Fan, Jun Podnar, Jessica W Wilcox, Edward Gipson, Jenny Gillaspy, Allison Jepsen, Kristen BonDurant, Sandra S Morris, Krystalynne Berkeley, Maura LeClerc, Ashley Simpson, Stephen D Sommerville, Gary Grimmett, Leslie Adams, Marie Butty, Vincent L G Levine, Stuart S. |
| author_sort | Herbert, Zachary T |
| collection | MIT |
| description | Background
Ribosomal RNA (rRNA) comprises at least 90% of total RNA extracted from mammalian tissue or cell line samples. Informative transcriptional profiling using massively parallel sequencing technologies requires either enrichment of mature poly-adenylated transcripts or targeted depletion of the rRNA fraction. The latter method is of particular interest because it is compatible with degraded samples such as those extracted from FFPE and also captures transcripts that are not poly-adenylated such as some non-coding RNAs. Here we provide a cross-site study that evaluates the performance of ribosomal RNA removal kits from Illumina, Takara/Clontech, Kapa Biosystems, Lexogen, New England Biolabs and Qiagen on intact and degraded RNA samples.
Results
We find that all of the kits are capable of performing significant ribosomal depletion, though there are differences in their ease of use. All kits were able to remove ribosomal RNA to below 20% with intact RNA and identify ~ 14,000 protein coding genes from the Universal Human Reference RNA sample at >1FPKM. Analysis of differentially detected genes between kits suggests that transcript length may be a key factor in library production efficiency.
Conclusions
These results provide a roadmap for labs on the strengths of each of these methods and how best to utilize them. Keywords: RNAseqr; RNA depletion; Illumina; NGS; ABRF; Transcriptomics |
| first_indexed | 2024-09-23T12:08:40Z |
| format | Article |
| id | mit-1721.1/114726 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T12:08:40Z |
| publishDate | 2018 |
| publisher | BioMed Central Ltd |
| record_format | dspace |
| spelling | mit-1721.1/1147262022-09-28T00:30:53Z Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction Herbert, Zachary T Kershner, Jamie P Thimmapuram, Jyothi Choudhari, Sulbha Alekseyev, Yuriy O Fan, Jun Podnar, Jessica W Wilcox, Edward Gipson, Jenny Gillaspy, Allison Jepsen, Kristen BonDurant, Sandra S Morris, Krystalynne Berkeley, Maura LeClerc, Ashley Simpson, Stephen D Sommerville, Gary Grimmett, Leslie Adams, Marie Butty, Vincent L G Levine, Stuart S. Massachusetts Institute of Technology. Department of Biology Butty, Vincent L G Levine, Stuart S. Background Ribosomal RNA (rRNA) comprises at least 90% of total RNA extracted from mammalian tissue or cell line samples. Informative transcriptional profiling using massively parallel sequencing technologies requires either enrichment of mature poly-adenylated transcripts or targeted depletion of the rRNA fraction. The latter method is of particular interest because it is compatible with degraded samples such as those extracted from FFPE and also captures transcripts that are not poly-adenylated such as some non-coding RNAs. Here we provide a cross-site study that evaluates the performance of ribosomal RNA removal kits from Illumina, Takara/Clontech, Kapa Biosystems, Lexogen, New England Biolabs and Qiagen on intact and degraded RNA samples. Results We find that all of the kits are capable of performing significant ribosomal depletion, though there are differences in their ease of use. All kits were able to remove ribosomal RNA to below 20% with intact RNA and identify ~ 14,000 protein coding genes from the Universal Human Reference RNA sample at >1FPKM. Analysis of differentially detected genes between kits suggests that transcript length may be a key factor in library production efficiency. Conclusions These results provide a roadmap for labs on the strengths of each of these methods and how best to utilize them. Keywords: RNAseqr; RNA depletion; Illumina; NGS; ABRF; Transcriptomics National Cancer Institute (U.S.) (Grant P30-CA14051) National Institute of Environmental Health Sciences (Grant P30-ES002109) 2018-04-13T19:22:24Z 2018-04-13T19:22:24Z 2018-03 2017-09 2018-03-18T04:12:03Z Article http://purl.org/eprint/type/JournalArticle 1471-2164 http://hdl.handle.net/1721.1/114726 Herbert, Zachary T. et al. "Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction" BMC Genomics 19, S2 (March 2018): 199 © 2018 The Author(s) en http://dx.doi.org/10.1186/s12864-018-4585-1 BMC Genomics Creative Commons Attribution http://creativecommons.org/licenses/by/4.0/ The Author(s). application/pdf BioMed Central Ltd BioMed Central |
| spellingShingle | Herbert, Zachary T Kershner, Jamie P Thimmapuram, Jyothi Choudhari, Sulbha Alekseyev, Yuriy O Fan, Jun Podnar, Jessica W Wilcox, Edward Gipson, Jenny Gillaspy, Allison Jepsen, Kristen BonDurant, Sandra S Morris, Krystalynne Berkeley, Maura LeClerc, Ashley Simpson, Stephen D Sommerville, Gary Grimmett, Leslie Adams, Marie Butty, Vincent L G Levine, Stuart S. Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction |
| title | Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction |
| title_full | Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction |
| title_fullStr | Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction |
| title_full_unstemmed | Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction |
| title_short | Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction |
| title_sort | cross site comparison of ribosomal depletion kits for illumina rnaseq library construction |
| url | http://hdl.handle.net/1721.1/114726 |
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