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A Guide for Designing and Analyzing RNA-Seq Data

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Gene Expression Analysis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1783))

Abstract

The identity of a cell or an organism is at least in part defined by its gene expression and therefore analyzing gene expression remains one of the most frequently performed experimental techniques in molecular biology. The development of the RNA-Sequencing (RNA-Seq) method allows an unprecedented opportunity to analyze expression of protein-coding, noncoding RNA and also de novo transcript assembly of a new species or organism. However, the planning and design of RNA-Seq experiments has important implications for addressing the desired biological question and maximizing the value of the data obtained. In addition, RNA-Seq generates a huge volume of data and accurate analysis of this data involves several different steps and choices of tools. This can be challenging and overwhelming, especially for bench scientists. In this chapter, we describe an entire workflow for performing RNA-Seq experiments. We describe critical aspects of wet lab experiments such as RNA isolation, library preparation and the initial design of an experiment. Further, we provide a step-by-step description of the bioinformatics workflow for different steps involved in RNA-Seq data analysis. This includes power calculations, setting up a computational environment, acquisition and processing of publicly available data if desired, quality control measures, preprocessing steps for the raw data, differential expression analysis, and data visualization. We particularly mention important considerations for each step to provide a guide for designing and analyzing RNA-Seq data.

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Acknowledgments

A.C. and M.R.E. are grateful to the New Zealand Institute for Cancer Research Trust for supporting their respective positions.

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Authors and Affiliations

  1. Department of Pathology, Dunedin School of Medicine, University of Otago, Dunedin, New Zealand

    Aniruddha Chatterjee, Antonio Ahn, Euan J. Rodger & Michael R. Eccles

  2. Maurice Wilkins Centre for Molecular Biodiscovery, Auckland, New Zealand

    Aniruddha Chatterjee, Euan J. Rodger & Michael R. Eccles

  3. Department of Biochemistry, University of Otago, Dunedin, New Zealand

    Peter A. Stockwell

Authors
  1. Aniruddha Chatterjee
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  2. Antonio Ahn
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  3. Euan J. Rodger
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  4. Peter A. Stockwell
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  5. Michael R. Eccles
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Corresponding author

Correspondence to Aniruddha Chatterjee .

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Editors and Affiliations

  1. Division of Geriatrics and Clinical Gerontology, National Institute on Aging, Bethesda, Maryland, USA

    Nalini Raghavachari

  2. Environmental Laboratory, US Army Engineer Research and Development Center, Vicksburg, Mississippi, USA

    Natàlia Garcia-Reyero

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Chatterjee, A., Ahn, A., Rodger, E.J., Stockwell, P.A., Eccles, M.R. (2018). A Guide for Designing and Analyzing RNA-Seq Data. In: Raghavachari, N., Garcia-Reyero, N. (eds) Gene Expression Analysis. Methods in Molecular Biology, vol 1783. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7834-2_3

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  • DOI: https://doi.org/10.1007/978-1-4939-7834-2_3

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7833-5

  • Online ISBN: 978-1-4939-7834-2

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