Michel Elena J S, Hotto Amber M, Strickler Susan R, Stern David B, Castandet Benoît
Boyce Thompson Institute, Ithaca, NY, USA.
Plant Biology Section, School of Integrative Plant Science, Cornell University, Ithaca NY, USA.
Methods Mol Biol. 2018;1829:295-313. doi: 10.1007/978-1-4939-8654-5_20.
Since its first use in plants in 2007, high-throughput RNA sequencing (RNA-Seq) has generated a vast amount of data for both model and nonmodel species. Organellar transcriptomes, however, are virtually always overlooked at the data analysis step. We therefore developed ChloroSeq, a bioinformatic pipeline aimed at facilitating the systematic analysis of chloroplast RNA metabolism, and we provide here a step-by-step user's manual. Following the alignment of quality-controlled data to the genome of interest, ChloroSeq measures genome expression level along with splicing and RNA editing efficiencies. When used in combination with the Tuxedo suite (TopHat and Cufflinks), ChloroSeq allows the simultaneous analysis of organellar and nuclear transcriptomes, opening the way to a better understanding of nucleus-organelle cross talk. We also describe the use of R commands to produce publication-quality figures based on ChloroSeq outputs. The effectiveness of the pipeline is illustrated through analysis of an RNA-Seq dataset covering the transition from growth to maturation to senescence of Arabidopsis thaliana leaves.
自2007年首次应用于植物以来，高通量RNA测序（RNA-Seq）已为模式生物和非模式生物生成了大量数据。然而，在数据分析步骤中，细胞器转录组几乎总是被忽视。因此，我们开发了ChloroSeq，这是一种旨在促进叶绿体RNA代谢系统分析的生物信息学流程，并且在此提供一份详细的用户手册。在将质量控制后的数据与目标基因组比对之后，ChloroSeq可测量基因组表达水平以及剪接和RNA编辑效率。当与Tuxedo套件（TopHat和Cufflinks）结合使用时，ChloroSeq能够同时分析细胞器和细胞核转录组，为更好地理解细胞核与细胞器之间的相互作用开辟了道路。我们还描述了如何使用R命令根据ChloroSeq的输出生成可用于发表的图表。通过对一个涵盖拟南芥叶片从生长到成熟再到衰老转变过程的RNA-Seq数据集进行分析，展示了该流程的有效性。
