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Systematic analysis of TruSeq, SMARTer and SMARTer Ultra-Low RNA-seq kits for standard, low and ultra-low quantity samples

High-throughput RNA-sequencing has become the gold standard method for whole-transcriptome gene expression analysis, and is widely used in numerous applications to study cell and tissue transcriptomes. It is also being increasingly used in a number of clinical applications, including expression prof...

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Autores principales: Palomares, Marie-Ange, Dalmasso, Cyril, Bonnet, Eric, Derbois, Céline, Brohard-Julien, Solène, Ambroise, Christophe, Battail, Christophe, Deleuze, Jean-François, Olaso, Robert
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6525156/
https://www.ncbi.nlm.nih.gov/pubmed/31101892
http://dx.doi.org/10.1038/s41598-019-43983-0
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author Palomares, Marie-Ange
Dalmasso, Cyril
Bonnet, Eric
Derbois, Céline
Brohard-Julien, Solène
Ambroise, Christophe
Battail, Christophe
Deleuze, Jean-François
Olaso, Robert
author_facet Palomares, Marie-Ange
Dalmasso, Cyril
Bonnet, Eric
Derbois, Céline
Brohard-Julien, Solène
Ambroise, Christophe
Battail, Christophe
Deleuze, Jean-François
Olaso, Robert
author_sort Palomares, Marie-Ange
collection PubMed
description High-throughput RNA-sequencing has become the gold standard method for whole-transcriptome gene expression analysis, and is widely used in numerous applications to study cell and tissue transcriptomes. It is also being increasingly used in a number of clinical applications, including expression profiling for diagnostics and alternative transcript detection. However, despite its many advantages, RNA sequencing can be challenging in some situations, for instance in cases of low input amounts or degraded RNA samples. Several protocols have been proposed to overcome these challenges, and many are available as commercial kits. In this study, we systematically test three recent commercial technologies for RNA-seq library preparation (TruSeq, SMARTer and SMARTer Ultra-Low) on human biological reference materials, using standard (1 mg), low (100 ng and 10 ng) and ultra-low (<1 ng) input amounts, and for mRNA and total RNA, stranded and unstranded. The results are analyzed using read quality and alignment metrics, gene detection and differential gene expression metrics. Overall, we show that the TruSeq kit performs well with an input amount of 100 ng, while the SMARTer kit shows decreased performance for inputs of 100 and 10 ng, and the SMARTer Ultra-Low kit performs relatively well for input amounts <1 ng. All the results are discussed in detail, and we provide guidelines for biologists for the selection of an RNA-seq library preparation kit.
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spelling pubmed-65251562019-05-29 Systematic analysis of TruSeq, SMARTer and SMARTer Ultra-Low RNA-seq kits for standard, low and ultra-low quantity samples Palomares, Marie-Ange Dalmasso, Cyril Bonnet, Eric Derbois, Céline Brohard-Julien, Solène Ambroise, Christophe Battail, Christophe Deleuze, Jean-François Olaso, Robert Sci Rep Article High-throughput RNA-sequencing has become the gold standard method for whole-transcriptome gene expression analysis, and is widely used in numerous applications to study cell and tissue transcriptomes. It is also being increasingly used in a number of clinical applications, including expression profiling for diagnostics and alternative transcript detection. However, despite its many advantages, RNA sequencing can be challenging in some situations, for instance in cases of low input amounts or degraded RNA samples. Several protocols have been proposed to overcome these challenges, and many are available as commercial kits. In this study, we systematically test three recent commercial technologies for RNA-seq library preparation (TruSeq, SMARTer and SMARTer Ultra-Low) on human biological reference materials, using standard (1 mg), low (100 ng and 10 ng) and ultra-low (<1 ng) input amounts, and for mRNA and total RNA, stranded and unstranded. The results are analyzed using read quality and alignment metrics, gene detection and differential gene expression metrics. Overall, we show that the TruSeq kit performs well with an input amount of 100 ng, while the SMARTer kit shows decreased performance for inputs of 100 and 10 ng, and the SMARTer Ultra-Low kit performs relatively well for input amounts <1 ng. All the results are discussed in detail, and we provide guidelines for biologists for the selection of an RNA-seq library preparation kit. Nature Publishing Group UK 2019-05-17 /pmc/articles/PMC6525156/ /pubmed/31101892 http://dx.doi.org/10.1038/s41598-019-43983-0 Text en © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as 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 images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Palomares, Marie-Ange
Dalmasso, Cyril
Bonnet, Eric
Derbois, Céline
Brohard-Julien, Solène
Ambroise, Christophe
Battail, Christophe
Deleuze, Jean-François
Olaso, Robert
Systematic analysis of TruSeq, SMARTer and SMARTer Ultra-Low RNA-seq kits for standard, low and ultra-low quantity samples
title Systematic analysis of TruSeq, SMARTer and SMARTer Ultra-Low RNA-seq kits for standard, low and ultra-low quantity samples
title_full Systematic analysis of TruSeq, SMARTer and SMARTer Ultra-Low RNA-seq kits for standard, low and ultra-low quantity samples
title_fullStr Systematic analysis of TruSeq, SMARTer and SMARTer Ultra-Low RNA-seq kits for standard, low and ultra-low quantity samples
title_full_unstemmed Systematic analysis of TruSeq, SMARTer and SMARTer Ultra-Low RNA-seq kits for standard, low and ultra-low quantity samples
title_short Systematic analysis of TruSeq, SMARTer and SMARTer Ultra-Low RNA-seq kits for standard, low and ultra-low quantity samples
title_sort systematic analysis of truseq, smarter and smarter ultra-low rna-seq kits for standard, low and ultra-low quantity samples
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6525156/
https://www.ncbi.nlm.nih.gov/pubmed/31101892
http://dx.doi.org/10.1038/s41598-019-43983-0
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