This repository contains all data, analysis code and documentation for the manuscript:

D-alanine Aminotransferase (Dat) promotes Staphylococcus aureus colonization fitness on human nasal respiratory epithelium

Reproducibility

All analyses are provided as individual Quarto (.qmd) notebooks, which can be rendered independently. Each notebook reads processed .rds files from the dataframes/ folder and generates publication-quality figures and statistical outputs (Table S2).

To reproduce the analyses:

• Clone or download this repository to your local machine.
• Download the dataframes/ and Tn-Seq/ folders and place it in your working directory.
• Download all .qmd notebooks from the repository and keep them in the same directory.
• Install required R packages listed in R Session Info using renv or manually.
• Render the notebooks using Quarto to generate figures and results.

You can set environment variable as follows in R:

Sys.setenv(BOX_PATH = "/path/to/your_project")

Alternatively, you can add this to your .Renviron file:

BOX_PATH=/path/to/your_project

The whole project can be rendered using RStudio's "Render" or "Build" options or in the terminal via:

quarto render

Repository Structure

This repository is organized as follows:

DatCol_project/
├── dataframes/          
│   ├── [input data / CSV tables]
│   ├── [input data / RDS files]
│   └── …
├── Tn-Seq/
│   ├── RefSeqs/
│   │   ├── [reference sequence files]
│   │   └── …
│   ├── sites/
│   │   ├── [insertion site files]
│   │   └── …
│   ├── scripts/
│   │   └── TnGeneBin.pl
└── [analysis notebooks saved as .qmd files]

Data folders

• dataframes/
  • Contains all processed data used to generate figures and statistical analyses (see Table S2)
  • Files are provided in .csv and .rds formats for easy visualization and reproducibility in R
  • .rds file names correspond to their associated .qmd analysis notebooks
  • DatCol_CFUs_HNO_Chemical.rds and DatCol_CFUs_HNO_Genetic.rds both correspond to the Complementation notebook
  • DatCol_CFUs_HNO_Controls.rds contains all monocolonization control data associated with HNO_Competition.qmd
• Tn-Seq/
  • Raw FASTQ files are available through the SRA [insert link or BioProject accession]
  • Processed insertion site files are included here to reproduce the Essential Gene Analysis and associated figure generation
  • Subfolders include:
    • RefSeqs/: reference genome files
    • sites/: transposon insertion site files
    • scripts/: includes the TnGeneBin.pl Perl script used to bin insertion sites for essential gene analysis

Analysis Notebooks

• Main Analysis Notebooks:
  • Tn-Seq Pipeline
  • Tn-Seq Essential Genes Analysis
  • Competition Assays on HNO-ALI
  • Complementation Assays on HNO-ALI
  • Growth Curves (OD600)
  • Cocolonization Assays with Corynebacterium pseudodiphtheriticum
  • Cytotoxicity Assays (LDH)
  • Alanine in HNO-ALI mucus
• Other Notebooks:
  • Linear Mixed Model Workflow
  • R Session Info
  • References