Categorical scatterplots with R for biologists: a step-by-step guide

Benjamin Petre1, Aurore Coince2, Sophien Kamoun1

1 The Sainsbury Laboratory, Norwich, UK; 2 Earlham Institute, Norwich, UK

Weissgerber and colleagues (2015) recently stated that ‘as scientists, we urgently need to change our practices for presenting continuous data in small sample size studies’. They called for more scatterplot and boxplot representations in scientific papers, which ‘allow readers to critically evaluate continuous data’ (Weissgerber et al., 2015). In the Kamoun Lab at The Sainsbury Laboratory, we recently implemented a protocol to generate categorical scatterplots (Petre et al., 2016; Dagdas et al., 2016). Here we describe the three steps of this protocol: 1) formatting of the data set in a .csv file, 2) execution of the R script to generate the graph, and 3) export of the graph as a .pdf file.

Protocol

• Step 1: format the data set as a .csv file. Store the data in a three-column excel file as shown in Powerpoint slide. The first column ‘Replicate’ indicates the biological replicates. In the example, the month and year during which the replicate was performed is indicated. The second column ‘Condition’ indicates the conditions of the experiment (in the example, a wild type and two mutants called A and B). The third column ‘Value’ contains continuous values. Save the Excel file as a .csv file (File -> Save as -> in ‘File Format’, select .csv). This .csv file is the input file to import in R.

• Step 2: execute the R script (see Notes 1 and 2). Copy the script shown in Powerpoint slide and paste it in the R console. Execute the script. In the dialog box, select the input .csv file from step 1. The categorical scatterplot will appear in a separate window. Dots represent the values for each sample; colors indicate replicates. Boxplots are superimposed; black dots indicate outliers.

• Step 3: save the graph as a .pdf file. Shape the window at your convenience and save the graph as a .pdf file (File -> Save as). See Powerpoint slide for an example.

Notes

• Note 1: install the ggplot2 package. The R script requires the package ‘ggplot2’ to be installed. To install it, Packages & Data -> Package Installer -> enter ‘ggplot2’ in the Package Search space and click on ‘Get List’. Select ‘ggplot2’ in the Package column and click on ‘Install Selected’. Install all dependencies as well.

• Note 2: use a log scale for the y-axis. To use a log scale for the y-axis of the graph, use the command line below in place of command line #7 in the script.

7 Display the graph in a separate window. Dot colors indicate

replicates

graph + geom_boxplot(outlier.colour='black', colour='black') + geom_jitter(aes(col=Replicate)) + scale_y_log10() + theme_bw()

References

Dagdas YF, Belhaj K, Maqbool A, Chaparro-Garcia A, Pandey P, Petre B, et al. (2016) An effector of the Irish potato famine pathogen antagonizes a host autophagy cargo receptor. eLife 5:e10856.

Petre B, Saunders DGO, Sklenar J, Lorrain C, Krasileva KV, Win J, et al. (2016) Heterologous Expression Screens in Nicotiana benthamiana Identify a Candidate Effector of the Wheat Yellow Rust Pathogen that Associates with Processing Bodies. PLoS ONE 11(2):e0149035

Weissgerber TL, Milic NM, Winham SJ, Garovic VD (2015) Beyond Bar and Line Graphs: Time for a New Data Presentation Paradigm. PLoS Biol 13(4):e1002128

https://cran.r-project.org/

http://ggplot2.org/

This database studies the performance inconsistency on the biomass HHV ultimate analysis. The research null hypothesis is the consistency in the rank of a biomass HHV model. Fifteen biomass models are trained and tested in four datasets. In each dataset, the rank invariability of these 15 models indicates the performance consistency.

The database includes the datasets and source codes to analyze the performance consistency of the biomass HHV. These datasets are stored in tabular on an excel workbook. The source codes are the biomass HHV machine learning model through the MATLAB Objected Orient Program (OOP). These machine learning models consist of eight regressions, four supervised learnings, and three neural networks.

An excel workbook, "BiomassDataSetUltimate.xlsx," collects the research datasets in six worksheets. The first worksheet, "Ultimate," contains 908 HHV data from 20 pieces of literature. The names of the worksheet column indicate the elements of the ultimate analysis on a % dry basis. The HHV column refers to the higher heating value in MJ/kg. The following worksheet, "Full Residuals," backups the model testing's residuals based on the 20-fold cross-validations. The article (Kijkarncharoensin & Innet, 2021) verifies the performance consistency through these residuals. The other worksheets present the literature datasets implemented to train and test the model performance in many pieces of literature.

A file named "SourceCodeUltimate.rar" collects the MATLAB machine learning models implemented in the article. The list of the folders in this file is the class structure of the machine learning models. These classes extend the features of the original MATLAB's Statistics and Machine Learning Toolbox to support, e.g., the k-fold cross-validation. The MATLAB script, name "runStudyUltimate.m," is the article's main program to analyze the performance consistency of the biomass HHV model through the ultimate analysis. The script instantly loads the datasets from the excel workbook and automatically fits the biomass model through the OOP classes.

The first section of the MATLAB script generates the most accurate model by optimizing the model's higher parameters. It takes a few hours for the first run to train the machine learning model via the trial and error process. The trained models can be saved in MATLAB .mat file and loaded back to the MATLAB workspace. The remaining script, separated by the script section break, performs the residual analysis to inspect the performance consistency. Furthermore, the figure of the biomass data in the 3D scatter plot, and the box plots of the prediction residuals are exhibited. Finally, the interpretations of these results are examined in the author's article.

Reference : Kijkarncharoensin, A., & Innet, S. (2022). Performance inconsistency of the Biomass Higher Heating Value (HHV) Models derived from Ultimate Analysis [Manuscript in preparation]. University of the Thai Chamber of Commerce.

This dataset includes all the raw micrographs, scatter-plots, cytometry data, graphs, excel sheets, and tomographic data used in the report entitled, "Rapid multi-well evaluation of assorted materials for hydrogel-assisted giant unilamellar vesicle production: empowering bottom-up synthetic biology."

Authors: Cherng-Wen Darren Tan1*, Magdalena Schöller2 and Eva-Kathrin Ehmoser3 1 University of Natural Resources and Life Sciences, Department of Bionanosciences, Institute of Synthetic Bioarchitectures, Muthgasse 11, Level 2, 1190, Vienna, Austria; darren.tan@boku.ac.at -mail.com2 University of Natural Resources and Life Sciences, Department of Bionanosciences, Institute of Synthetic Bioarchitectures, Muthgasse 11, Level 2, 1190, Vienna, Austria; magdalena.schoeller@boku.ac.at3 University of Natural Resources and Life Sciences, Department of Bionanosciences, Institute of Synthetic Bioarchitectures, Muthgasse 11, Level 2, 1190, Vienna, Austria; eva.ehmoser@boku.ac.at *Correspondence: darren.tan@boku.ac.at -mail.com; Tel.: +43 1 47654-80457