Abstract
Sequencing costs are falling, but the cost of data analysis remains high, often because unforeseen problems arise, such as insufficient depth of sequencing or batch effects. Experimenting with data analysis methods during the planning phase of an experiment can reveal unanticipated problems and build valuable bioinformatics expertise in the organism or process being studied. This protocol describes using R Markdown and RStudio, user-friendly tools for statistical analysis and reproducible research in bioinformatics, to analyze and document the analysis of an example RNA-Seq data set from tomato pollen undergoing chronic heat stress. Also, we show how to use Integrated Genome Browser to visualize read coverage graphs for differentially expressed genes. Applying the protocol described here and using the provided data sets represent a useful first step toward building RNA-Seq data analysis expertise in a research group.
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Acknowledgements
The example data set was from the Workshop in Next-Generation Sequencing (WiNGS), which was co-sponsored by the NSF Research Coordination Network on Integrative Pollen Biology (award 0955431), the NSF Plant Genome Research Program (award 1238051), and the Department of Bioinformatics and Genomics at UNC Charlotte. NIH R01 grant number 21737838 supports development of the IGB software.
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Loraine, A.E., Blakley, I.C., Jagadeesan, S., Harper, J., Miller, G., Firon, N. (2015). Analysis and Visualization of RNA-Seq Expression Data Using RStudio, Bioconductor, and Integrated Genome Browser. In: Alonso, J., Stepanova, A. (eds) Plant Functional Genomics. Methods in Molecular Biology, vol 1284. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2444-8_24
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DOI: https://doi.org/10.1007/978-1-4939-2444-8_24
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