title: Influence of RNA extraction methods and library selection schemes on RNA-seq data.
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.
authors.affiliation: Max Planck Institute for Molecular Genetics, Ihnestr, 63-73, Berlin 14195, Germany. sultan.marc@gmail.com.
authors.forename: Marc
authors.initials: M
authors.lastname: Sultan
authors.forename: Vyacheslav
authors.initials: V
authors.lastname: Amstislavskiy
authors.forename: Thomas
authors.initials: T
authors.lastname: Risch
authors.forename: Moritz
authors.initials: M
authors.lastname: Schuette
authors.forename: Simon
authors.initials: S
authors.lastname: Dökel
authors.forename: Meryem
authors.initials: M
authors.lastname: Ralser
authors.forename: Daniela
authors.initials: D
authors.lastname: Balzereit
authors.forename: Hans
authors.initials: H
authors.lastname: Lehrach
authors.forename: Marie-Laure
authors.initials: ML
authors.lastname: Yaspo
mesh_terms: D020869:Gene Expression Profiling; D015723:Gene Library; D057809:HEK293 Cells; D006801:Humans; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D012326:RNA Splicing; D062085:RNA, Long Noncoding / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*; D017423:Sequence Analysis, RNA; D059467:Transcriptome
chemical_list: D062085:RNA, Long Noncoding; D012333:RNA, Messenger; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf
references.citation: Nature. 1985 Nov 28-Dec 4;318(6044):371-4
references.pmid: 2415825
references.citation: PLoS One. 2011 May 06;6(5):e19287
references.pmid: 21573114
references.citation: BMC Genomics. 2012 Sep 24;13:504
references.pmid: 23006825
references.citation: Nat Methods. 2008 Jul;5(7):621-8
references.pmid: 18516045
references.citation: Cell. 2009 Feb 20;136(4):629-41
references.pmid: 19239885
references.citation: Chromosoma. 2007 Aug;116(4):373-83
references.pmid: 17333237
references.citation: Nat Struct Mol Biol. 2011 Nov 06;18(12):1435-40
references.pmid: 22056773
references.citation: Genes Dev. 1989 Dec;3(12B):2172-9
references.pmid: 2576416
references.citation: Methods Mol Biol. 2010;667:93-111
references.pmid: 20827529
references.citation: Genome Res. 2011 Dec;21(12):2213-23
references.pmid: 21903743
references.citation: Bioinformatics. 2009 Jul 15;25(14):1754-60
references.pmid: 19451168
references.citation: Nat Genet. 2008 Dec;40(12):1413-5
references.pmid: 18978789
references.citation: Genes Dev. 2011 Sep 15;25(18):1915-27
references.pmid: 21890647
references.citation: Proc Natl Acad Sci U S A. 2009 Jul 14;106(28):11667-72
references.pmid: 19571010
references.citation: Nucleic Acids Res. 2010 Jun;38(10):e112
references.pmid: 20150413
references.citation: Cell. 2013 Jul 3;154(1):26-46
references.pmid: 23827673
references.citation: Science. 2005 Sep 2;309(5740):1564-6
references.pmid: 16141073
references.citation: Genomics. 2010 Nov;96(5):259-65
references.pmid: 20688152
references.citation: Nucleic Acids Res. 2012 Aug;40(14):6391-400
references.pmid: 22492512
references.citation: Nucleic Acids Res. 2011 Oct;39(18):e120
references.pmid: 21737426
references.citation: Science. 2008 Aug 15;321(5891):956-60
references.pmid: 18599741
references.citation: PLoS Biol. 2010 May 18;8(5):e1000371
references.pmid: 20502517
references.citation: Biochem Biophys Res Commun. 2012 Jun 15;422(4):643-6
references.pmid: 22609201
references.citation: Nature. 2012 Sep 6;489(7414):101-8
references.pmid: 22955620
references.citation: Blood. 2010 Dec 2;116(23):e118-27
references.pmid: 20733160
references.citation: Nature. 1991 Jan 3;349(6304):38-44
references.pmid: 1985261
references.citation: PLoS One. 2008 Jul 30;3(7):e2803
references.pmid: 18665230
references.citation: Mol Cell Biol. 2008 Sep;28(18):5583-94
references.pmid: 18625719
references.citation: Ann N Y Acad Sci. 2005 Nov;1058:196-204
references.pmid: 16394137
references.citation: Genome Biol. 2011;12(2):R16
references.pmid: 21324177
references.citation: EMBO Rep. 2009 Aug;10(8):894-900
references.pmid: 19575011
references.citation: Nucleic Acids Res. 1990 Jan 11;18(1):97-103
references.pmid: 2308839
references.citation: PLoS Biol. 2011 Jul;9(7):e1000625; discussion e1001102
references.pmid: 21765801
references.citation: BMC Genomics. 2012 Oct 30;13:574
references.pmid: 23110385
references.citation: Cell. 1983 Feb;32(2):461-72
references.pmid: 6186397
references.citation: Mol Cell. 2007 May 11;26(3):349-65
references.pmid: 17499042
references.citation: Genome Biol. 2013 Apr 25;14(4):R36
references.pmid: 23618408
references.citation: BMC Biol. 2010 Dec 21;8:149
references.pmid: 21176148
references.citation: Genome Biol. 2010;11(7):R78
references.pmid: 20653958
references.citation: Science. 2005 May 20;308(5725):1149-54
references.pmid: 15790807
references.citation: J Biol Chem. 2005 May 20;280(20):19721-7
references.pmid: 15769737
references.citation: BMC Genomics. 2009 Jun 12;10:264
references.pmid: 19523228
references.citation: Nature. 2009 Mar 12;458(7235):223-7
references.pmid: 19182780
references.citation: RNA. 2009 Oct;15(10):1917-28
references.pmid: 19703940
references.citation: Bioinformatics. 2010 Mar 15;26(6):841-2
references.pmid: 20110278
references.citation: Mol Cell Biol. 2005 Aug;25(15):6427-35
references.pmid: 16024781
references.citation: FASEB J. 2000 Feb;14(2):242-54
references.pmid: 10657981