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  Influence of RNA extraction methods and library selection schemes on RNA-seq data

Sultan, M., Amstislavskiy, V., Risch, T., Schütte, M., Dökel, S., Ralser, M., et al. (2014). Influence of RNA extraction methods and library selection schemes on RNA-seq data. BMC Genomics, 15: 15:675. doi:10.1186/1471-2164-15-675.

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© 2014 Sultan et al
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Sultan, Marc1, Author           
Amstislavskiy, Vyacheslav1, Author           
Risch, Thomas1, Author           
Schütte, Moritz1, Author           
Dökel, Simon1, Author
Ralser, Meryem1, Author
Balzereit, Daniela1, Author           
Lehrach, Hans2, Author           
Yaspo, Marie Laure3, Author           
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1Human Chromosome 21 (Marie-Laure Yaspo), Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society, ou_1479652              
2Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society, ou_1433550              
3Gene Regulation and Systems Biology of Cancer (Marie-Laure Yaspo), Independent Junior Research Groups (OWL), Max Planck Institute for Molecular Genetics, Max Planck Society, ou_2117287              

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 Abstract: BACKGROUND: Gene expression analysis by RNA sequencing is now widely used in a number of applications surveying the whole transcriptomes of cells and tissues. The recent introduction of ribosomal RNA depletion protocols, such as RiboZero, has extended the view of the polyadenylated transcriptome to the poly(A)- fraction of the RNA. However, substantial amounts of intronic transcriptional activity has been reported in RiboZero protocols, raising issues regarding their potential nuclear origin and the impact on the actual sequence depth in exonic regions. RESULTS: Using HEK293 human cells as source material, we assessed here the impact of the two commonly used RNA extraction methods and of the library construction protocols (rRNA depletion versus mRNA) on 1) the relative abundance of intronic reads and 2) on the estimation of gene expression values. We benchmarked the rRNA depletion-based sequencing with a specific analysis of the cytoplasmic and nuclear transcriptome fractions, suggesting that the large majority of the intronic reads correspond to unprocessed nuclear transcripts rather than to independent transcriptional units. We show that Qiagen or TRIzol extraction methods retain differentially nuclear RNA species, and that consequently, rRNA depletion-based RNA sequencing protocols are particularly sensitive to the extraction methods. CONCLUSIONS: We could show that the combination of Trizol-based RNA extraction with rRNA depletion sequencing protocols led to the largest fraction of intronic reads, after the sequencing of the nuclear transcriptome. We discuss here the impact of the various strategies on gene expression and alternative splicing estimation measures. Further, we propose guidelines and a double selection strategy for minimizing the expression biases, without loss of information.

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Language(s): eng - English
 Dates: 2014-08-11
 Publication Status: Published online
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 Rev. Type: Peer
 Identifiers: DOI: 10.1186/1471-2164-15-675
ISSN: 1471-2164 (Electronic)
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Title: BMC Genomics
Source Genre: Journal
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Publ. Info: BioMed Central
Pages: - Volume / Issue: 15 Sequence Number: 15:675 Start / End Page: - Identifier: ISSN: 1471-2164
CoNE: https://pure.mpg.de/cone/journals/resource/111000136905010