English
 
User Manual Privacy Policy Disclaimer Contact us
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT
  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.

Item is

Basic

show hide
Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0025-B633-D Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0025-B634-B
Genre: Journal Article

Files

show Files
hide Files
:
Sultan.pdf (Publisher version), 2MB
Name:
Sultan.pdf
Description:
-
Visibility:
Public
MIME-Type / Checksum:
application/pdf / [MD5]
Technical Metadata:
Copyright Date:
-
Copyright Info:
© 2014 Sultan et al
License:
-

Locators

show
hide
Description:
-

Creators

show
hide
 Creators:
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              
Affiliations:
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              

Content

show
hide
Free keywords: -
 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.

Details

show
hide
Language(s): eng - English
 Dates: 2014-08-11
 Publication Status: Published online
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1186/1471-2164-15-675
ISSN: 1471-2164 (Electronic)
 Degree: -

Event

show

Legal Case

show

Project information

show

Source 1

show
hide
Title: BMC Genomics
Source Genre: Journal
 Creator(s):
Affiliations:
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