Transcriptomics of Wood Frog and American Bullfrog Bd Response

This repository contains code, data, and figures that support:

Eskew, E.A., B.C. Shock, E.E.B. LaDouceur, K. Keel, M.R. Miller, J.E. Foley, and B.D. Todd. 2018. Gene expression differs in susceptible and resistant amphibians exposed to Batrachochytrium dendrobatidis. Royal Society Open Science 5: 170910.

The primary aim of this project was to help elucidate the mechanisms by which some amphibian host species are resistant to or tolerant of the fungal pathogen Batrachochytrium dendrobatidis (Bd) given that infection in many species results in high mortality, leading to population declines and species-level extinctions. Thus, we experimentally exposed two common amphibian species expected to differ in disease response, wood frogs and American bullfrogs, to Bd and measured a suite of outcomes. This repository provides data and code related to frog survival, body mass change, and Bd infection prevalence and load (evaluated via both qPCR and histology). Ultimately, these data support the key product from our work: an RNA sequencing dataset generated from ventral skin tissues from 87 study animals. RNA-seq samples were collected from both amphibian host species at multiple time points following Bd exposure. After sequencing and read quality processing, reads were mapped to an existing amphibian reference transcriptome using Salmon. This repository includes Salmon data resulting from two alternative read processing pipelines: 1) processing using a combination of ConDeTri and Trimmomatic or 2) processing via the expHTS pipeline. Ultimately, we use the latter data for downstream analyses. Differential gene expression analyses were conducted using the edgeR package.

Raw sequence data for this project are available at NCBI's Sequence Read Archive accession number SRP111327.

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Repository Structure

• data\ contains all data files necessary to conduct the analyses
  • annotation\ subdirectory contains gene annotation data used in bioinformatics_analyses.R
  • expHTS_preprocess\ subdirectory contains metadata resulting from expHTS processing of raw RNA-seq reads
  • salmon\ subdirectory contains read count data (generated from the Salmon software) used in bioinformatics_analyses.R
• figures\ contains all figures that are output from analysis scripts
• R\ contains code for functions that are sourced in the analysis scripts
• scripts\ contains all scripts necessary to recreate this project's analyses
  • bioinformatics_analyses.R contains code for RNA-seq data analyses
  • body_measurement_analyses.R contains code for frog body mass and length analyses
  • expHTS_preprocess_analyses.R contains code for plotting of expHTS metadata and calculation of post-processing RNA-seq read counts
  • histology_analyses.R contains code for analyses comparing infection quantification with histology and qPCR
  • infection_analyses.R contains code for infection prevalence and load analyses (using qPCR data)
  • survival_analyses.R contains code for frog survival analyses

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Reproducing the Analyses

Assuming you have all the required R packages, recreating analyses and figures is as simple as running the relevant script from the scripts directory. Scripts are written such that figures will be output to the figures directory. Note that some scripts, particuarly bioinformatics_analyses.R, will take quite a while to work through.

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License

This project is licensed under the terms of the MIT license.