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/

In "Sample Student Data", there are 6 sheets. There are three sheets with sample datasets, one for each of the three different exercise protocols described (CrP Sample Dataset, Glycolytic Dataset, Oxidative Dataset). Additionally, there are three sheets with sample graphs created using one of the three datasets (CrP Sample Graph, Glycolytic Graph, Oxidative Graph). Each dataset and graph pairs are from different subjects. · CrP Sample Dataset and CrP Sample Graph: This is an example of a dataset and graph created from an exercise protocol designed to stress the creatine phosphate system. Here, the subject was a track and field athlete who threw the shot put for the DeSales University track team. The NIRS monitor was placed on the right triceps muscle, and the student threw the shot put six times with a minute rest in between throws. Data was collected telemetrically by the NIRS device and then downloaded after the student had completed the protocol. · Glycolytic Dataset and Glycolytic Graph: This is an example of a dataset and graph created from an exercise protocol designed to stress the glycolytic energy system. In this example, the subject performed continuous squat jumps for 30 seconds, followed by a 90 second rest period, for a total of three exercise bouts. The NIRS monitor was place on the left gastrocnemius muscle. Here again, data was collected telemetrically by the NIRS device and then downloaded after he had completed the protocol. · Oxidative Dataset and Oxidative Graph: In this example, the dataset and graph are from an exercise protocol designed to stress the oxidative system. Here, the student held a sustained, light-intensity, isometric biceps contraction (pushing against a table). The NIRS monitor was attached to the left biceps muscle belly. Here, data was collected by a student observing the SmO2 values displayed on a secondary device; specifically, a smartphone with the IPSensorMan APP displaying data. The recorder student observed and recorded the data on an Excel Spreadsheet, and marked the times that exercise began and ended on the Spreadsheet.

The two datasets contained in the repository have all necessary information to reproduce the results reported in the paper. Actors&9E_full.csv: affiliation matrix of actors and water management plans. The values in the matrix are 1 (if the actor participated in the plan) and 0 otherwise. Node_attributes: dataset containing all variables used in the models reported in the paper.