WholeLungvBALFluid

The purpose of this repository is to provide public access to the R code and data files used in the following publication:

Baker JM, Hinkle KJ, McDonald RA, Brown CA, Falkowski NR, Huffnagle GB, Dickson RP. Whole lung tissue is the preferred sampling method for amplicon-based characterization of murine lung microbiota. Microbiome 9, 99 (2021). https://doi.org/10.1186/s40168-021-01055-4

Outline of Analytical Approach for Low-Biomass Specimens

1. Specimen collection and processing

• Goal: Selection of appropriate numbers and types of negative and positive controls - sample all potential source communities
• Method: Thoughtful experimental design and sterile specimen collection

2. Quantification

• Goal: Confirm sufficient quantities of DNA (bacterial and total) have beeen isolated from low-biomass samples
• Method: droplet digital PCR or qPCR for bacterial DNA quantification, Qubit or Nanodrop for total DNA quantification

3. Sequencing, Identification, and Quantification of Bacterial Reads

• Goal: Amplify, sequence, identify, and quantify bacterial reads in low-biomass specimens
• Method: PCR amplification, 16S rRNA gene amplicon sequencing, mothur, calculation of read counts per specimen

4. Diversity of Identified Bacterial Taxa

• Goal: Determine bacterial signal is distinct from controls and reproducible among biological replicates
• Method: Calculation of alpha (e.g. rarified richness, Shannon diversity index) and beta (e.g. Bray-Curtis dissimilarity index) diversity metrics

5. Relative Abundance of Identified Bacterial Taxa

• Goal: Determine the biological plausibility of bacterial taxa identified in low-biomass samples
• Method: Principal component analysis and ranked relative abundance of bacterial taxa

Description of Files included in Repository

• Rmarkdown with code (defaults to final dataset version)
• Taxonomy file (contains taxonomy assignments for each OTU from mothur)
• Shared file (contains OTU counts from mothur)
• 16S rRNA gene ddPCR quantification data
• Read count data from MiSeq run
• Knitted reports (raw html and pdf) for untrimmed, trimmed, and final datasets for comparison
• PDF versions of each figure generated for the final publication, & additional figures that did not make the final publication
• License information
• Hidden files (.RData and .Rhistory from local sessions)
• .gitignore file (ignores cache and other hidden files when pushing directory contents to GitHub)

External Links:

• Link to publication: https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-021-01055-4
• Link to preprint: https://www.biorxiv.org/content/10.1101/2020.09.08.283259v1
• Link to SRA submission: https://www.ncbi.nlm.nih.gov/sra/PRJNA644805
• Links to Rmarkdown htmls:
  • Trimmed & Quality-Checked Dataset (final version):
    • Development Link: https://raw.githack.com/jennbaker95/WholeLungvBALFluid/dev/WholeLung_v_BALF_Code_trimmedQCfinal.html
    • Permanent Link: https://rawcdn.githack.com/jennbaker95/WholeLungvBALFluid/cd54d5ec864ea7a90e905a3c2fae51de67d8f406/WholeLung_v_BALF_Code_trimmedQCfinal.html
  • Untrimmed dataset:
    • Development link: https://raw.githack.com/jennbaker95/WholeLungvBALFluid/dev/WholeLung_v_BALF_Code_untrimmed.html
    • Permanent Link: https://rawcdn.githack.com/jennbaker95/WholeLungvBALFluid/cee2674080f51aedb26c9dfe4a8cde38737e052e/WholeLung_v_BALF_Code_untrimmed.html
  • Trimmed dataset:
    • Development Link: https://raw.githack.com/jennbaker95/WholeLungvBALFluid/dev/WholeLung_v_BALF_Code_trimmed.html
    • Permanent Link: https://rawcdn.githack.com/jennbaker95/WholeLungvBALFluid/cee2674080f51aedb26c9dfe4a8cde38737e052e/WholeLung_v_BALF_Code_trimmed.html

Packages and dependencies required to run code in the repository can be found in session info in the knitted Rmarkdown pdf and html.