Plastids pp 295-313 | Cite as

A Guide to the Chloroplast Transcriptome Analysis Using RNA-Seq

  • Elena J. S. Michel
  • Amber M. Hotto
  • Susan R. Strickler
  • David B. Stern
  • Benoît Castandet
Part of the Methods in Molecular Biology book series (MIMB, volume 1829)


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.

Key words

RNA-Seq ChloroSeq Chloroplast Organelles Leaf development 



This work was supported by Grant DE-FG02-10ER20015 from the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences, of the US Department of Energy. The authors would also like to thank the Boyce Thompson Institute Bioinformatics Help Desk for assistance with experimental methods. This is a consulting service for early-stage ideas or issues in bioinformatics. The IPS2 benefits from the support of the LabEx Saclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS).


  1. 1.
    Ozsolak F, Milos PM (2011) RNA sequencing: advances, challenges and opportunities. Nat Rev Genet 12(2):87–98. Scholar
  2. 2.
    van Dijk EL, Auger H, Jaszczyszyn Y et al (2014) Ten years of next-generation sequencing technology. Trends Genet 30(9):418–426. Scholar
  3. 3.
    Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10(1):57–63. Scholar
  4. 4.
    Trapnell C, Williams BA, Pertea G et al (2010) Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 28(5):511–515. Scholar
  5. 5.
    Ghosh S, Chan CK (2016) Analysis of RNA-Seq data using TopHat and cufflinks. Methods Mol Biol 1374:339–361. Scholar
  6. 6.
    Trapnell C, Roberts A, Goff L et al (2012) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and cufflinks. Nature Protoc 7(3):562–578. Scholar
  7. 7.
    Goecks J, Nekrutenko A, Taylor J (2010) Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol 11(8):R86. Scholar
  8. 8.
    Ding Y, Tang Y, Kwok CK et al (2014) In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features. Nature 505(7485):696–700. Scholar
  9. 9.
    Gosai SJ, Foley SW, Wang D et al (2015) Global analysis of the RNA-protein interaction and RNA secondary structure landscapes of the Arabidopsis nucleus. Mol Cell 57(2):376–388. Scholar
  10. 10.
    Zheng Q, Ryvkin P, Li F et al (2010) Genome-wide double-stranded RNA sequencing reveals the functional significance of base-paired RNAs in Arabidopsis. PLoS Genet 6(9):e1001141. Scholar
  11. 11.
    Wu X, Liu M, Downie B et al (2011) Genome-wide landscape of polyadenylation in Arabidopsis provides evidence for extensive alternative polyadenylation. Proc Natl Acad Sci U S A 108(30):12533–12538. Scholar
  12. 12.
    Chotewutmontri P, Barkan A (2016) Dynamics of chloroplast translation during chloroplast differentiation in maize. PLoS Genet 12(7):e1006106. Scholar
  13. 13.
    Juntawong P, Girke T, Bazin J et al (2014) Translational dynamics revealed by genome-wide profiling of ribosome footprints in Arabidopsis. Proc Natl Acad Sci U S A 111(1):E203–E212. Scholar
  14. 14.
    Liu MJ, Wu SH, Wu JF et al (2013) Translational landscape of photomorphogenic Arabidopsis. Plant Cell 25(10):3699–3710. Scholar
  15. 15.
    Di C, Yuan J, Wu Y et al (2014) Characterization of stress-responsive lncRNAs in Arabidopsis thaliana by integrating expression, epigenetic and structural features. Plant J 80(5):848–861. Scholar
  16. 16.
    Bombarely A, Edwards KD, Sanchez-Tamburrino J et al (2012) Deciphering the complex leaf transcriptome of the allotetraploid species Nicotiana tabacum: a phylogenomic perspective. BMC Genomics 13:406. Scholar
  17. 17.
    Smith DR (2013) RNA-Seq data: a goldmine for organelle research. Brief Funct Genomics 12(5):454–456. Scholar
  18. 18.
    Gros F, Hiatt H, Gilbert W et al (1961) Unstable ribonucleic acid revealed by pulse labelling of Escherichia coli. Nature 190:581–585CrossRefPubMedGoogle Scholar
  19. 19.
    Jacob F, Monod J (1961) Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol 3:318–356CrossRefPubMedGoogle Scholar
  20. 20.
    Ycas M, Vincent WS (1960) A ribonucleic acid fraction from yeast related in composition to desoxyribonucleic acid. Proc Natl Acad Sci U S A 46(6):804–811CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Cui P, Lin Q, Ding F et al (2010) A comparison between ribo-minus RNA-sequencing and polyA-selected RNA-sequencing. Genomics 96(5):259–265. Scholar
  22. 22.
    Lange H, Sement FM, Canaday J et al (2009) Polyadenylation-assisted RNA degradation processes in plants. Trends Plant Sci 14(9):497–504. Scholar
  23. 23.
    Rorbach J, Bobrowicz A, Pearce S et al (2014) Polyadenylation in bacteria and organelles. Meth Mol Biol 1125:211–227. Scholar
  24. 24.
    Castandet B, Hotto AM, Strickler SR et al (2016) ChloroSeq, an optimized chloroplast RNA-Seq bioinformatic pipeline, reveals remodeling of the organellar transcriptome under heat stress. G3 (Bethesda). Scholar
  25. 25.
    Zhao W, He X, Hoadley KA et al (2014) Comparison of RNA-Seq by poly (A) capture, ribosomal RNA depletion, and DNA microarray for expression profiling. BMC Genomics 15:419. Scholar
  26. 26.
    Barkan A (2011) Expression of plastid genes: organelle-specific elaborations on a prokaryotic scaffold. Plant Physiol 155(4):1520–1532. Scholar
  27. 27.
    Germain A, Hotto AM, Barkan A et al (2013) RNA processing and decay in plastids. Wiley Interdiscip Rev RNA 4(3):295–316. Scholar
  28. 28.
    Stern DB, Goldschmidt-Clermont M, Hanson MR (2010) Chloroplast RNA metabolism. Annu Rev Plant Biol 61:125–155. Scholar
  29. 29.
    Hotto AM, Castandet B, Gilet L et al (2015) Arabidopsis chloroplast mini-ribonuclease III participates in rRNA maturation and intron recycling. Plant Cell 27(3):724–740. Scholar
  30. 30.
    Castandet B, Hotto AM, Fei Z et al (2013) Strand-specific RNA sequencing uncovers chloroplast ribonuclease functions. FEBS Lett 587(18):3096–3101. Scholar
  31. 31.
    Germain A, Herlich S, Larom S et al (2011) Mutational analysis of Arabidopsis chloroplast polynucleotide phosphorylase reveals roles for both RNase PH core domains in polyadenylation, RNA 3'-end maturation and intron degradation. Plant J 67(3):381–394. Scholar
  32. 32.
    Hotto AM, Schmitz RJ, Fei Z et al (2011) Unexpected diversity of chloroplast noncoding RNAs as revealed by deep sequencing of the arabidopsis transcriptome. G3 (Bethesda) 1(7):559–570. Scholar
  33. 33.
    Walter M, Kilian J, Kudla J (2002) PNPase activity determines the efficiency of mRNA 3'-end processing, the degradation of tRNA and the extent of polyadenylation in chloroplasts. EMBO J 21(24):6905–6914CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Smith DR, Sanita Lima M (2016) Unraveling chloroplast transcriptomes with ChloroSeq, an organelle RNA-Seq bioinformatics pipeline. Brief Bioinform.
  35. 35.
    Chan KX, Phua SY, Crisp P et al (2016) Learning the languages of the chloroplast: retrograde signaling and beyond. Annu Rev Plant Biol 67:25–53. Scholar
  36. 36.
    Quinlan AR (2014) BEDTools: the Swiss-army tool for genome feature analysis. Curr Protoc Bioinformatics 47:11.12.1–34. doi: Scholar
  37. 37.
    Li H, Handsaker B, Wysoker A et al (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25(16):2078–2079. Scholar
  38. 38.
    Woo HR, Koo HJ, Kim J et al (2016) Programming of plant leaf senescence with temporal and inter-organellar coordination of transcriptome in Arabidopsis. Plant Physiol 171(1):452–467. Scholar
  39. 39.
    Boussardon C, Salone V, Avon A et al (2012) Two interacting proteins are necessary for the editing of the NdhD-1 site in Arabidopsis plastids. Plant Cell 24(9):3684–3694. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Elena J. S. Michel
    • 1
    • 3
  • Amber M. Hotto
    • 1
  • Susan R. Strickler
    • 1
  • David B. Stern
    • 1
  • Benoît Castandet
    • 1
    • 2
    • 4
  1. 1.Boyce Thompson InstituteIthacaUSA
  2. 2.Centre National de la Recherche Scientifique, Institute of Plant Sciences Paris Saclay, Institut National de la Recherche AgronomiqueUniversité Paris-Sud, Université Evry, Université Paris-SaclayOrsayFrance
  3. 3.Plant Biology Section, School of Integrative Plant ScienceCornell UniversityIthaca NYUSA
  4. 4.Institute of Plant Sciences Paris-Saclay IPS2Paris Diderot, Sorbonne Paris-CitéFrance

Personalised recommendations