RT Journal
A1 Hammer, Paul
A1 Banck, Michaela S.
A1 Amberg, Ronny
A1 Wang, Cheng
A1 Petznick, Gabriele
A1 Luo, Shujun
A1 Khrebtukova, Irina
A1 Schroth, Gary P.
A1 Beyerlein, Peter
A1 Beutler, Andreas S.
T1 mRNA-seq with agnostic splice site discovery for nervous system transcriptomics tested in chronic pain
JF Genome Research 
JO Genome Research 
YR 2010 
FD June 01 
VO 20 
IS 6 
SP 847 
OP 860 
DO 10.1101/gr.101204.109 
UL http://genome.cshlp.org/content/20/6/847.abstract 
AB 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.