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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 targeted depletion of...
Autores principales: | , , , , , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6389247/ https://www.ncbi.nlm.nih.gov/pubmed/29703133 http://dx.doi.org/10.1186/s12864-018-4585-1 |
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author | Herbert, Zachary T. Kershner, Jamie P. Butty, Vincent L. Thimmapuram, Jyothi Choudhari, Sulbha Alekseyev, Yuriy O. Fan, Jun Podnar, Jessica W. Wilcox, Edward Gipson, Jenny Gillaspy, Allison Jepsen, Kristen BonDurant, Sandra Splinter Morris, Krystalynne Berkeley, Maura LeClerc, Ashley Simpson, Stephen D. Sommerville, Gary Grimmett, Leslie Adams, Marie Levine, Stuart S. |
author_facet | Herbert, Zachary T. Kershner, Jamie P. Butty, Vincent L. Thimmapuram, Jyothi Choudhari, Sulbha Alekseyev, Yuriy O. Fan, Jun Podnar, Jessica W. Wilcox, Edward Gipson, Jenny Gillaspy, Allison Jepsen, Kristen BonDurant, Sandra Splinter Morris, Krystalynne Berkeley, Maura LeClerc, Ashley Simpson, Stephen D. Sommerville, Gary Grimmett, Leslie Adams, Marie Levine, Stuart S. |
author_sort | Herbert, Zachary T. |
collection | PubMed |
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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12864-018-4585-1) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-6389247 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-63892472019-03-19 Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction Herbert, Zachary T. Kershner, Jamie P. Butty, Vincent L. Thimmapuram, Jyothi Choudhari, Sulbha Alekseyev, Yuriy O. Fan, Jun Podnar, Jessica W. Wilcox, Edward Gipson, Jenny Gillaspy, Allison Jepsen, Kristen BonDurant, Sandra Splinter Morris, Krystalynne Berkeley, Maura LeClerc, Ashley Simpson, Stephen D. Sommerville, Gary Grimmett, Leslie Adams, Marie Levine, Stuart S. BMC Genomics Methodology Article 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12864-018-4585-1) contains supplementary material, which is available to authorized users. BioMed Central 2018-03-15 /pmc/articles/PMC6389247/ /pubmed/29703133 http://dx.doi.org/10.1186/s12864-018-4585-1 Text en © The Author(s). 2018 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Methodology Article Herbert, Zachary T. Kershner, Jamie P. Butty, Vincent L. Thimmapuram, Jyothi Choudhari, Sulbha Alekseyev, Yuriy O. Fan, Jun Podnar, Jessica W. Wilcox, Edward Gipson, Jenny Gillaspy, Allison Jepsen, Kristen BonDurant, Sandra Splinter Morris, Krystalynne Berkeley, Maura LeClerc, Ashley Simpson, Stephen D. Sommerville, Gary Grimmett, Leslie Adams, Marie 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 |
topic | Methodology Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6389247/ https://www.ncbi.nlm.nih.gov/pubmed/29703133 http://dx.doi.org/10.1186/s12864-018-4585-1 |
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