RNA-Seq data processing and gene expression analysis

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Reproducible Research

In any experiment where computation plays a critical role in generating the results and conclusions, researchers should ensure that the presentation of their work includes reproducibility, meaning “the ability to recompute data analytic results given an observed dataset and knowledge of the data analysis pipeline.”¹. This is distinct from the concept of replicability, in which “the chance that an independent experiment targeting the same scientific question will produce a consistent result”¹.

For a RNA-Seq experiment, reproducible means documenting in detail all the steps taken from the original fastq files through the end of the statistical analysis and any downstream data mining. The reasons to do this are multifold, three of which are: 1) so reviewers can assess whether the computational steps are valid and match what was described in the report, 2) to serve as a record for your lab, a “computational” notebook equivalent to the laboratory notebook and 3) to serve as a teaching guide for colleagues to use in other experiments² (see Introduction). The typical Methods section allowed in a publication only gives a brief description of the steps taken and is not sufficient for reproducibility. Instead, the documentation for reproducibility can go into supplementary files and typically includes codes for software calls and parameters, statistical analysis and generation of figures and tables for the publication. Advanced users can create versioned-controlled Rmarkdown-type documents that integrate codes with figures, graphs and written explanations² (see Tools) but simple README.txt and AnalysisCodes.R files can also suffice. Beware to limit the manual manipulation of intermediate files, which is difficult to describe and hence difficult to reproduce. Instead, the output files from one section should be the input files for the next section, or else manually-created files should be put in the supplemental as well.

Finally, note that reproducible research documentation is addition to, and not a replacement for, the written report that describes the reasons behind the selected choices and synthesizes the overall understanding and issues involved in RNA-Seq analysis.

For more information and guidance, see:

• Leek & Peng 2015¹
• rOpenSci’s Reproducibilty Guide²
• Peng 2011³
• Peng 2014 blog post at SimplyStatistics.org

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