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Automatically format Gene ontology data for graphical representation with R

Prerequisites for MacOS

Install Xcode from the App Store (it is quite long)

image-20210607125457492

Start Xcode to allow complete installation. The computer pass word will be required.

Quit.

Prerequisites for Windows

Install UNIX terminal

  • Download on your PC the file TerminalUnixSetup.exe

    This is an executable that will install a UNIX terminal in your HOME directory

  • Execute TerminalUnixSetup.exe

    The UNIX terminal comes with all necessary tools. A tools directory is created in your HOME. It contains the script prepare_gene_onthology.pl

Lancement du terminal

Select Git Bash in the Windows menu or click the icon on your desk.

image-20210405175008398

Script for data preparation

Install file on MacOS

Preparation

  • Save the file prepare_gene_onthology.pl in Downloads

  • Open a UNIX terminal : Terminal

    image-20210422085133689

Create a tools directory that will contain the script

  • Execute the following command

    mkdir -p $HOME/tools

Install the prepare_gene_onthology.pl file in the tools directory

  • Execute the following commandsto move the file and make it executable

    mv $HOME/Downloads/prepare_gene_onthology.pl $HOME/tools
chmod +x $HOME/tools/prepare_gene_onthology.pl

  • Close the terminal.

Install file on Windows

The file was already install by the executable TerminalUnixSetup.exe 😄

First execution

Open a UNIX terminal in the directory that contains the file to analyse

Start the script prepare_gene_onthology.pl

$HOME/tools/prepare_gene_onthology.pl

If the terminal shows the message below you can jump to the "Next executions" part.

prepare_gene_onthology.pl [--help|--man|--version]

or

prepare_gene_onthology.pl [-m|--method] [-c|--correction] input_gene_list.tsv output_curated_gene_ontology.tsv

On the contrary, if you get the message below, that means that some Perl modules need to be installed :

Au moins un des modules Perl nécessaires n'est pas installé.
Pour utiliser ce script vous devez d'abord exécuter les commandes suivantes:

cpan App::cpanminus
cpanm WWW::Mechanize
cpanm JSON

Create or update.zshrc file

touch .zshrc .bashrc

Execute command:

cpan App::cpanminus

This command may ask questions.

Accept all default answer with Enter

⚠️ ​**==Open a new tab in the terminal with==** cmd+t

Then execute:

cpanm WWW::Mechanize

This step can take some times, be patient

Finally, execute :

cpanm JSON

Next executions

The script prepare_gene_onthology.pl perform gene ontology analysis using PANTHER and REVIGO from a gene ID list according to the protocole descibed by Bonnot et al, 2019 . It formats the result so that it can be used by the scripts script_1plot.R and script_2plot.R which perform graphical representation of the ontology analysis.

PANTHER now, sort the results in a graphical order, so that each GO_id is link to the so called parent or main GO_id that characterize a class of ontology. This information is kept in the file output_go_ids_hierarchy.tsv and is used by the script script_1plot_hierarchy.R.

⚠️ The main GO_id whne alone don’t show up in the list but are took into account by de R script

The command is :

$HOME/tools/prepare_gene_onthology.pl [-m|--method] [-c|--correction] input_gene_list.tsv output_curated_gene_ontology.tsv output_go_ids_hierarchy.tsv

The script has 2 option : [-m|--method] and [-c|--correction] .

The value for the [-m|--method]are : biological_process cellular_component molecular_function

Default value is biological_process.

The value for [-c|--correction] are : fdr

​ bonferroni

Default value is fdr.

input_gene_list.tsv is the file that contains your data, let's call it myfile.tsv for the exemple.

The extention.tsv means "Tab-separated values". Any file with the data in tab separated columns is suitable to use with the script, no matter of the extention (a .txt file can be used).

output_curated_gene_ontology.tsv is the default name for the output file, you can change it..

Command can be :

$HOME/tools/prepare_gene_onthology.pl --method biological_process --correction fdr myfile.tsv my_output_file.tsv my_hierarchy_file.tsv

Or :

$HOME/tools/prepare_gene_onthology.pl -m biological_process -c fdr myfile.tsv my_output_file.tsv my_hierarchy_file.tsv

Get syntax information with :

prepare_gene_onthology.pl --man

You get the manual. To close it press q.

If you execute the command:

$HOME/tools/prepare_gene_onthology.pl --method biological_process --correction fdr myfile.tsv my_output_file.tsv my_hierarchy_file.tsv

Terminal shows the below message :

Step 1/7 Extract gene ID list from /Volumes/Disk_4To/Donnees_ARA2/Clusters/2w_5clusters/cluster1.txt
Step 2/7 Panther ontology analysis => /tmp/gene_ontology_analysis.txt, /tmp/gene_ontology_analysis.json
request...
export result to /tmp/gene_ontology_analysis.txt
export result to /tmp/gene_ontology_analysis.json
Step 3/7 extract GO ids and FDR from /tmp/gene_ontology_analysis.txt
Step 4/7 REVIGO reduction => /tmp/gene_ontology_analysis_revigo.csv
request...
export result
Step 5/7 Formating /Users/cecile/Downloads/output_curated_gene_ontology.tsv: filter panther result with revigo result
Step 6/7 keep track of GO ids hierarchy from /tmp/gene_ontology_analysis.json into /Users/cecile/Downloads/output_go_ids_hierarchy.tsv
Step 7/7 cleanup: remove temporay files from /tmp

⚠️ Access to REVIGO (step 4/7) can be long, even too long. If the script exit with an error of type: "request.Error POSTing http://revigo.irb.hr/QueryJobStatus.aspx: read timeout...", re-start the same command as often as necessary.

Graphical representation of the results

The 2 scripts script_1plot.R and script_2plot.R allows to plot respectively one graph or 2 graph in the same figure.

The third script script_1plot_hierarchy.R take into account the hierarchical informations.

The scripts can be executed in R-studio. All scripts check package requirement and install only the missing ones.

Comments in the scripts explain each step.

At the end of the scripts, the command :

InfoSession <- devtools::session_info()

# Save session file
 write.table(InfoSession, file = "InfoSession.txt", 
                quote = FALSE, row.names = FALSE, sep = '\t')

Allows you to keep information about R session (version of R, R-studio, packages...) to comply with FAIR analysis guide line.

Citations

PANTHER

PANTHER version 16: a revised family classification, tree-based classification tool, enhancer regions and extensive API Huaiyu Mi, Dustin Ebert, Anushya Muruganujan, Caitlin Mills, Laurent-Philippe Albou, Tremayne Mushayamaha and Paul D Thomas . Nucl. Acids Res. (2020) doi: 10.1093/nar/gkaa1106s.

REVIGO

Supek F, Bošnjak M, Škunca N, Šmuc T. "REVIGO summarizes and visualizes long lists of Gene Ontology terms" PLoS ONE 2011. doi:10.1371/journal.pone.0021800

Script R

A Simple Protocol for Informative Visualization of Enriched Gene Ontology Terms. T. Bonnot, MB. Gillard and DH. Nagel. Bio-101: e3429. DOI:10.21769/BioProtoc.3429

R Packages

R Core Team. 2020. R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing. https://www.R-project.org/.

Wickham, Hadley. 2016. Ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York. https://ggplot2.tidyverse.org.

Wickham, Hadley, Jim Hester, and Winston Chang. 2021. Devtools: Tools to Make Developing r Packages Easier. https://CRAN.R-project.org/package=devtools.

Wilke, Claus O. 2020. Cowplot: Streamlined Plot Theme and Plot Annotations for ’Ggplot2’. https://CRAN.R-project.org/package=cowplot.

Script prepare_gene_onthology.pl

Terese M. et Lecampion C. https://github.com/cecile-lecampion/gene-ontology-analysis-and-graph

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