title: mRNA-seq with agnostic splice site discovery for nervous system transcriptomics tested in chronic pain.
abstract: mRNA-seq is a paradigm-shifting technology because of its superior sensitivity and dynamic range and its potential to capture transcriptomes in an agnostic fashion, i.e., independently of existing genome annotations. Implementation of the agnostic approach, however, has not yet been fully achieved. In particular, agnostic mapping of pre-mRNA splice sites has not been demonstrated. The present study pursued dual goals: (1) to advance mRNA-seq bioinformatics toward unbiased transcriptome capture and (2) to demonstrate its potential for discovery in neuroscience by applying the approach to an in vivo model of neurological disease. We have performed mRNA-seq on the L4 dorsal root ganglion (DRG) of rats with chronic neuropathic pain induced by spinal nerve ligation (SNL) of the neighboring (L5) spinal nerve. We found that 12.4% of known genes were induced and 7% were suppressed in the dysfunctional (but anatomically intact) L4 DRG 2 wk after SNL. These alterations persisted chronically (2 mo). Using a read cluster classifier with strong test characteristics (ROC area 97%), we discovered 10,464 novel exons. A new algorithm for agnostic mapping of pre-mRNA splice junctions (SJs) achieved a precision of 97%. Integration of information from all mRNA-seq read classes including SJs led to genome reannotations specifically relevant for the species used (rat), the anatomical site studied (DRG), and the neurological disease considered (pain); for example, a 64-exon coreceptor for the nociceptive transmitter substance P was identified, and 21.9% of newly discovered exons were shown to be dysregulated. Thus, mRNA-seq with agnostic analysis methods appears to provide a highly productive approach for in vivo transcriptomics in the nervous system.
authors.affiliation: University of Applied Sciences, Wildau 15745, Germany.
authors.forename: Paul
authors.initials: P
authors.lastname: Hammer
authors.forename: Michaela S
authors.initials: MS
authors.lastname: Banck
authors.forename: Ronny
authors.initials: R
authors.lastname: Amberg
authors.forename: Cheng
authors.initials: C
authors.lastname: Wang
authors.forename: Gabriele
authors.initials: G
authors.lastname: Petznick
authors.forename: Shujun
authors.initials: S
authors.lastname: Luo
authors.forename: Irina
authors.initials: I
authors.lastname: Khrebtukova
authors.forename: Gary P
authors.initials: GP
authors.lastname: Schroth
authors.forename: Peter
authors.initials: P
authors.lastname: Beyerlein
authors.forename: Andreas S
authors.initials: AS
authors.lastname: Beutler
mesh_terms: D000818:Animals; D002908:Chronic Disease; D005727:Ganglia, Spinal / Q000378:metabolism*; D020869:Gene Expression Profiling; D010146:Pain / Q000235:genetics*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats
chemical_list: D012333:RNA, Messenger
references.citation: Nat Genet. 2008 Dec;40(12):1413-5
references.pmid: 18978789
references.citation: Science. 2005 Sep 2;309(5740):1564-6
references.pmid: 16141073
references.citation: Nature. 2008 Jun 26;453(7199):1239-43
references.pmid: 18488015
references.citation: Nat Methods. 2008 Jan;5(1):19-21
references.pmid: 18165803
references.citation: Trends Genet. 2005 Feb;21(2):93-102
references.pmid: 15661355
references.citation: Nature. 2008 Nov 27;456(7221):470-6
references.pmid: 18978772
references.citation: Genome Res. 2008 Sep;18(9):1509-17
references.pmid: 18550803
references.citation: Nature. 2008 Nov 6;456(7218):53-9
references.pmid: 18987734
references.citation: Genome Biol. 2003;4(2):105
references.pmid: 12620110
references.citation: Nat Methods. 2008 Jul;5(7):621-8
references.pmid: 18516045
references.citation: J Neurochem. 2003 Nov;87(3):560-73
references.pmid: 14535940
references.citation: BMC Neurosci. 2002 Aug 20;3:11
references.pmid: 12188929
references.citation: Science. 2008 Aug 15;321(5891):956-60
references.pmid: 18599741
references.citation: Genome Biol. 2009;10(3):R25
references.pmid: 19261174
references.citation: Nature. 2007 Jun 14;447(7146):799-816
references.pmid: 17571346
references.citation: J Neurosci. 2002 Feb 15;22(4):1303-15
references.pmid: 11850458
references.citation: Methods Mol Med. 2004;99:35-45
references.pmid: 15131327
references.citation: BMC Neurosci. 2002 Oct 25;3:16
references.pmid: 12401135
references.citation: Nature. 2009 Feb 5;457(7230):741-4
references.pmid: 19092807
references.citation: Nat Methods. 2008 Jul;5(7):613-9
references.pmid: 18516046
references.citation: Proc Natl Acad Sci U S A. 2008 Jan 22;105(3):1055-60
references.pmid: 18215993
references.citation: Science. 2008 Jun 6;320(5881):1344-9
references.pmid: 18451266
references.citation: Nat Rev Genet. 2009 Jan;10(1):57-63
references.pmid: 19015660
references.citation: Bioinformatics. 2009 May 1;25(9):1105-11
references.pmid: 19289445
references.citation: Curr Opin Mol Ther. 2005 Oct;7(5):431-9
references.pmid: 16248278
references.citation: Genome Res. 2010 Jan;20(1):45-58
references.pmid: 19858364
references.citation: PLoS One. 2008;3(11):e3625
references.pmid: 18985160
references.citation: Science. 2007 Jun 8;316(5830):1497-502
references.pmid: 17540862
references.citation: Genome Res. 2009 Jun;19(6):1044-56
references.pmid: 19273619
references.citation: Nat Biotechnol. 2006 Sep;24(9):1151-61
references.pmid: 16964229
references.citation: Nat Med. 2006 Nov;12(11):1269-77
references.pmid: 17057711
references.citation: J Physiol Pharmacol. 2006 Sep;57(3):401-14
references.pmid: 17033093