mRNA-seq with agnostic splice site discovery for nervous system transcriptomics tested in chronic pain
- Paul Hammer1,6,
- Michaela S. Banck2,6,
- Ronny Amberg1,2,6,
- Cheng Wang3,7,
- Gabriele Petznick1,4,
- Shujun Luo5,
- Irina Khrebtukova5,
- Gary P. Schroth5,
- Peter Beyerlein1,4 and
- Andreas S. Beutler2,8
- 1 University of Applied Sciences, Wildau 15745, Germany;
- 2 Mayo Clinic, Rochester, Minnesota 55905, USA;
- 3 Mount Sinai School of Medicine, New York, New York 10029, USA;
- 4 Philips Research Laboratories, Eindhoven 5656AE, Netherlands;
- 5 Illumina Inc., Hayward, California 94545, USA
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↵6 These authors contributed equally to this work.
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.
Footnotes
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↵8 Corresponding author.
E-mail beutler.andreas{at}mayo.edu.
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[Supplemental material is available online at http://www.genome.org. Sequence reads from this study have been submitted to the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under accession no. GSE20895. Software is available for download from http://www.th-wildau.de/bioinformatics/wios/ and http://mayoresearch.mayo.edu/mayo/research/beutler_lab/wios.cfm.]
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Article published online before print. Article and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.101204.109.
- Received September 27, 2009.
- Accepted March 23, 2010.
- Copyright © 2010 by Cold Spring Harbor Laboratory Press
Freely available online through the Genome Research Open Access option.