mRNA-seq with agnostic splice site discovery for nervous system transcriptomics tested in chronic pain
mRNA-seq with agnostic splice site discovery for nervous system transcriptomics tested in chronic pain
复制标题
DOI：
10.1101/gr.101204.109
复制
发表时间：
2010-06-01
期刊：
影响因子：
7
通讯作者：
Beutler, Andreas S.
中科院分区：
文献类型：
--
作者：
Hammer, Paul;Banck, Michaela S.;Beutler, Andreas S.
MRNA-SEQ是一种范式转换技术，因为它具有优越的灵敏度和动态范围，并且有可能以一种不可知的方式捕获转录本，即独立于现有的基因组注释。然而，不可知论方法的实施尚未完全实现。特别是，前信使核糖核酸剪接位点的不可知性图谱尚未得到证实。目前的研究追求双重目标：(1)推动mRNA-SEQ生物信息学朝着无偏见的转录组捕获方向发展；(2)通过将该方法应用于神经系统疾病的活体模型，展示其在神经科学中发现的潜力。我们对腰5脊神经结扎(SNL)所致慢性神经病理性痛大鼠的L4背根神经节(DRG)进行了mRNA-SEQ检测。我们发现，SNL后2周，12.4%的已知基因在功能障碍(但解剖完整)的L4DRG中被诱导，7%被抑制。这些改变持续存在(2个月)。使用具有很强测试特征(ROC面积为97%)的Read聚类分类器，我们发现了10,464个新的外显子。一种新的前mRNA剪接连接(SJS)不可知定位算法达到了97%的精度。整合了包括SJS在内的所有mna-seq阅读类的信息，导致了与所用物种(大鼠)、研究的解剖部位(DRG)和被考虑的神经疾病(疼痛)特定相关的基因组重新注释；例如，发现了伤害性递质P物质的外显子共受体，新发现的外显子中有21.9%被证明是调控不良的。因此，带有不可知性分析方法的mRNA-seq似乎为神经系统的体内转录提供了一种高效的方法。
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.