Skip to main content

Barley (Hordeum Vulgare) Anther and Meiocyte RNA Sequencing: Mapping Sequencing Reads and Downstream Data Analyses

  • Protocol
  • First Online:
Plant Gametogenesis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2484))

Abstract

RNA sequencing (RNA-seq) data is by now the most common method to study differential gene expression. Here we present a pipeline from RNA-seq generation to analysis with examples based on our own barley anther and meiocyte transcriptome. The bioinformatics pipeline will give everyone, from a beginner to a more experienced user, the possibility to analyze their datasets and identify significantly differentially expressed genes. It also allows differential alternative splicing analysis which will become increasingly common due to the high regulatory impact on the gene expression. We describe use of the Galaxy interface for RNA-seq read quantification and the 3D RNA-seq app for the downstream data analysis.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Geniza M, Jaiswal P (2017) Tools for building de novo transcriptome assembly. Curr Plant Biol 11-12:41–45

    Article  Google Scholar 

  2. Hölzer M, Marz M (2019) De novo transcriptome assembly: a comprehensive cross-species comparison of short-read RNA-Seq assemblers. GigaScience 8(5):giz039

    Article  Google Scholar 

  3. Lamarre S, Frasse P, Zouine M et al (2018) Optimization of an RNA-Seq differential gene expression analysis depending on biological replicate number and library size. Front Plant Sci 9:108

    Article  Google Scholar 

  4. Clark S, Yu F, Gu L et al (2019) Expanding alternative splicing identification by integrating multiple sources of transcription data in tomato. Front Plant Sci 10:689

    Article  Google Scholar 

  5. Zhang R, Calixto CPG, Marquez Y et al (2017) A high quality Arabidopsis transcriptome for accurate transcript-level analysis of alternative splicing. Nucleic Acids Res 45(9):5061–5073

    Article  CAS  Google Scholar 

  6. Barakate A, Orr J, Schreiber M et al (2021) Barley anther and Meiocyte transcriptome dynamics in meiotic prophase I. Front Plant Sci 11:619404

    Article  Google Scholar 

  7. Leinonen R, Sugawara H, Shumway M et al (2011) The sequence read archive. Nucleic Acids Res 39(Database issue):D19–D21

    Article  CAS  Google Scholar 

  8. Afgan E, Baker D, Batut B et al (2018) The galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res 46(W1):W537–W544

    Article  CAS  Google Scholar 

  9. Guo W, Tzioutziou NA, Stephen G et al (2020) 3D RNA-seq: a powerful and flexible tool for rapid and accurate differential expression and alternative splicing analysis of RNA-seq data for biologists. RNA Biol 18(11):1574–1587

    Google Scholar 

  10. Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30(15):2114–2120

    Article  CAS  Google Scholar 

  11. Patro R, Duggal G, Love MI et al (2017) Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods 14(4):417–419

    Article  CAS  Google Scholar 

  12. Srivastava A, Malik L, Smith T et al (2019) Alevin efficiently estimates accurate gene abundances from dscRNA-seq data. Genome Biol 20(1):65

    Article  Google Scholar 

  13. R Core Team. (2020) R: a language and environment for statistical computing. https://www.R-project.org/

  14. Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26(1):139–140

    Article  CAS  Google Scholar 

  15. Risso D, Ngai J, Speed TP et al (2014) Normalization of RNA-seq data using factor analysis of control genes or samples. Nat Biotechnol 32(9):896–902

    Article  CAS  Google Scholar 

  16. Ritchie ME, Phipson B, Wu D et al (2015) Limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res 43(7):e47

    Article  Google Scholar 

  17. Guo W, Calixto CPG, Brown JWS et al (2017) TSIS: an R package to infer alternative splicing isoform switches for time-series data. Bioinformatics 33(20):3308–3310

    Article  CAS  Google Scholar 

  18. Alexa A, Rahnenfuhrer J. (2020) topGO: Enrichment Analysis for Gene Ontology. R package version 2.46.0. https://bioconductor.org/packages/release/bioc/html/topGO.html

  19. Walter W, Sanchez-Cabo F, Ricote M (2015) GOplot: an R package for visually combining expression data with functional analysis. Bioinformatics 31(17):2912–2914

    Article  CAS  Google Scholar 

  20. Wickham H et al (2019) Welcome to the tidyverse. J Open Source Soft 4(43):1686. https://doi.org/10.21105/joss.01686

    Article  Google Scholar 

  21. Gehlenborg N (2019). UpSetR: a more scalable alternative to Venn and Euler diagrams for visualizing intersecting sets. R package version 1.4.0. https://CRAN.R-project.org/package=UpSetR

  22. Brunson JC and Read QD (2020). ggalluvial: Alluvial Plots in “ggplot2”. R package version 0.12.3. http://corybrunson.github.io/ggalluvial/

  23. Chen H (2018). VennDiagram: generate high-resolution Venn and Euler plots. R package version 1.6.20. https://CRAN.R-project.org/package=VennDiagram

  24. Yan L (2021). ggvenn: Draw Venn Diagram by 'ggplot2'. R package version 0.1.8. https://CRAN.R-project.org/package=ggvenn

  25. Gao C (2019). ggVennDiagram: A “ggplot2” implement of Venn diagram. R package version 0.3. https://CRAN.R-project.org/package=ggVennDiagram

  26. Wilkinson L (2011). venneuler: Venn and Euler Diagrams. R package version 1.1-0. https://CRAN.R-project.org/package=venneuler

  27. Larsson J (2020). _eulerr: area-proportional Euler and Venn diagrams with ellipses. R package version 6.1.0. https://cran.r-project.org/package=eulerr

  28. Chaudhari AK, Chaudhary BR (2012) Meiotic chromosome behaviour and karyomorphology of Aloe vera (L.) Burm. f. Chromosom Bot 7(1):23–29

    Article  Google Scholar 

  29. Colas I, Darrier B, Arrieta M et al (2017) Observation of extensive chromosome Axis remodeling during the “diffuse-phase” of meiosis in large genome cereals. Front Plant Sci 8:1235

    Article  Google Scholar 

  30. Gómez JF, Wilson ZA (2012) Non-destructive staging of barley reproductive development for molecular analysis based upon external morphology. J Exp Bot 63(11):4085–4094

    Article  Google Scholar 

  31. Huerta-Cepas J, Szklarczyk D, Heller D et al (2019) eggNOG 5.0: a hierarchical, functionally and phylogenetically annotated orthology resource based on 5090 organisms and 2502 viruses. Nucleic Acids Res 47(D1):D309–D314

    Article  CAS  Google Scholar 

  32. Törönen P, Medlar A, Holm L (2018) PANNZER2: a rapid functional annotation web server. Nucleic Acids Res 46(W1):W84–W88

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Robbie Waugh .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Schreiber, M., Orr, J., Barakate, A., Waugh, R. (2022). Barley (Hordeum Vulgare) Anther and Meiocyte RNA Sequencing: Mapping Sequencing Reads and Downstream Data Analyses. In: Lambing, C. (eds) Plant Gametogenesis. Methods in Molecular Biology, vol 2484. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2253-7_20

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2253-7_20

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2252-0

  • Online ISBN: 978-1-0716-2253-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics