DMA of D. magna - Paper - Data Analysis Code

This repository provides the R code to reproduce the data analysis summaries and figures for the Deep Metabolome Annotation (DMA) of Daphnia magna paper.

Overview

The repository contains three main analysis workflows:

1. Daphnia annotation summary - Analysis of metabolite annotations from D. magna samples
2. Metabolite reference standards analysis summary - Analysis of metabolite standard mixture (MSM) data
3. Phylo analysis - Phylogenetic/metabolomics analysis across species
4. Example Feature Check (Galaxy workflow history access) - Example for how the Galaxy workflow histories can be investigated

Project Structure

├── input/
│   ├── input_for_feature_check/          # Inputs for Galaxy workflow feature check
│   │   ├── galaxy_peaklist_references.csv
│   │   └── GalaxyNone-[samplelist_dma_daphnia_magna.tabular].tabular
│   ├── input_for_summary_plots/          # Data for Daphnia and MSM analysis
│   │   ├── merged_annotations_all_classified.zip
│   │   ├── metabolite_standard_mixture_details.csv
│   │   └── pubchem_set.zip
│   └── input_for_phylometab_plot/        # Data for phylometab analysis
│       ├── chebi_with_inchikey_source_classyfire.csv
│       ├── Daphnia_ChEBI.csv
│       ├── MTox.csv
│       ├── phyloT_generated_tree_1734701763_newick.txt
│       └── pubchem_kegg_hmdb_expanded.zip
├── output/                               # Generated figures and summary tables
├── example_feature_check.R               # Galaxy workflow feature check example
├── paper_summarise_daphnia.R             # Main Daphnia annotation analysis
├── paper_summarise_msm.R                 # Metabolite standard mixture analysis
└──  paper_phylometab.R                    # Phylometab metabolomics analysis

Requirements

• R (>= 4.4.3)
• RStudio (recommended)
• Required R packages are managed via renv (see Installation section)

Installation

1. Clone this repository
2. Open the R project in RStudio: dmagna-dma-paper.Rproj
3. init the R environment using renv:

renv::init()

This will install all required packages with their exact versions as specified in renv.lock.

Usage

1. Daphnia Annotation Analysis

Run the main Daphnia annotation summarization:

source("paper_summarise_daphnia.R")

Generates:

• Summary statistics and visualizations of metabolite annotations
• Classification analysis (superclass, class, subclass)
• Workflow comparison plots
• Venn diagrams for extraction methods, chromatography types, and polarity
• PCA analysis of annotations
• Tree maps and upset plots

Key outputs:

• FIG_5a_tree_map.pdf - Tree map visualization
• FIG_5b_annotations_all_pca.pdf - PCA plot of annotations
• FIG_5c-e_*_bar.pdf - Bar charts for chemical classifications
• FIG_6a-e_*.pdf - Workflow and method comparison plots
• daphnia_annotation_summary.csv - Summary statistics table
• FIG_27-29.pdf/png - Supplementary annotation summary plots

2. Metabolite Standard Mixture Analysis

Run the metabolite reference standards analysis:

source("paper_summarise_msm.R")

Generates:

• Analysis of metabolite standard mixture (MSM) annotations
• Workflow-specific analysis for MSM data

Key outputs:

• FIG_S30a_galaxy_msms_workflow_bar.pdf - MSM workflow analysis
• FIG_S30b_treemap_msm.pdf - MSM tree map
• FIG_S31_presence_absence_match_type_msm.pdf - Match type analysis
• msm_annotations_summary.csv - MSM summary statistics

3. Phylo Analysis

Run the phylogenetic/ metabolomics analysis:

source("paper_phylometab.R")

Generates:

• Phylogenetic tree with metabolite presence/absence data
• Cross-species metabolite comparison
• Database mapping analysis (KEGG, HMDB, MTox, ChEBI)

Key output:

• FIG_7_phylomet.pdf - Phylogenetic metabolomics plot

4. Example Feature Check (Galaxy workflow history access)

Use the example feature check to show how readers can directly access files from Galaxy workflows and verify LC-MS feature details against blank-filtered XCMS features.

source("example_feature_check.R")

What it does:

• Downloads XCMS peak lists and xcmsSet objects from Galaxy URLs
• Rebuilds RT windows and performs blank filtering
• Links the XCMS features from the Galaxy workflow to full annotation list

Inputs:

• Galaxy workflow file URLs in input/input_for_feature_check/galaxy_peaklist_references.csv
• Sample metadata in input/input_for_feature_check/GalaxyNone-[samplelist_dma_daphnia_magna.tabular].tabular
• Full annotation list in input/input_for_summary_plots/merged_annotations_all_classified.zip

Key outputs (per assay in output/<assay_name>/):

• *_DE_blank_filtered.RDS and *_blank_filtered_peak_matrix.csv
• *_xcms_passed_annos.csv

Key Dependencies

The analysis relies on several R packages:

• Data manipulation: dplyr, tidyr, data.table, stringr
• Visualization: ggplot2, cowplot, treemap, VennDiagram, UpSetR
• Chemical informatics: ChemmineR
• Phylogenetics: ape, ggtree, aplot
• Data import: openxlsx, jsonlite

Figures Generated

The code reproduces the following figures from the paper:

Main Figures:

• Figure 5: Metabolite annotation overview (tree map, PCA, classification bars)
• Figure 6: Workflow and method comparisons (Venn diagrams, upset plots)
• Figure 7: Phylogenetic metabolomics analysis

Supplementary Figures:

• Aditional method comparisons and MSM analysis

Output Files

All generated figures are saved as PDF files in the output/ directory. Summary tables are saved as CSV files for further analysis or inclusion in manuscripts.

Also includes an updated metabolites file created for the MetaboLights study MTBLS2273.

License

See LICENSE file for details.

Citation

If you use this code or data within this repo please cite the corresponding D. magna DMA paper.