Overview Tomato-Rhizosphere-Microbiome-Selection

Code for analysis and data visualization of tomato rhizosphere microbiome selection experiment. Code files included for 1) insect performance and plant trait data from three separate selection experiments that used a generalist and two specialist insects and 2) metabarcoding analysis (16s & ITS) of tomato rhizosphere soil microbiomes collected across 6 generations of a selection experiment from plants infested with aphids or uninfested control plants.

This repository contains all the code necessary to replicate the data analysis and figure generation for the manuscript "Plant-guided microbiome selection produces transient effects on insect performance and rhizosphere community assembly " authors: Laramy Enders, Elizabeth French, MacKenzie Kjeldgaard, Emily Tronson, and Ian Kaplan. {UPDATE w/ REFERENCE & LINK when published}

Insect Performance and Plant Trait Analysis

All data files containing insect performance measures and plant traits are located in the data folder of this respository - 1) data from two specialist insects used in selection experiments (M.euphorbidae, M.sexta) is located in file "Specialist.Insect.Data_Gens1_10.csv" 2) data from generalist insect used in experiments (S.exigua) is located in file "Spodoptera.All_Data_Gens1_6.csv".

To replicate data analysis and generation of figures run the "RhizoSelection_Trait_Analysis.Rmd" file.

Metabarcoding Analysis (16S & ITS)

To replicate this analysis, first download raw sequence data from NCBI's SRA database, BioProject number {INSERT}. Then run the "16S_master.trimmer.sub" and "ITS_master.trimmer.sub" scripts to trim illumina adapters and remove primer sequences.

Once these steps have been completed, run "dada2_16S_preprocessing.Rmd" and "dada2_ITS_preprocessing.Rmd" codes to perform dada2 preprocessing steps (alignment, error correction, chimera removal, taxonomy assignment, non-target seq removal, contaminant removal). These files are located in the code folder of this repository.

Silva (16S) and UNITE (ITS) taxonomic databases used - make sure these databases have been updated to the latest version. Download the necessary files to complete preprocessing steps. Silva: https://www.arb-silva.de/browser/ UNITE (select General FASTA release): https://unite.ut.ee/repository.php

Then run "RhizoSelection_16S_Analysis.Rmd" and "RhizoSelection_ITS_Analysis.Rmd" files to perform final data analysis and generate figures for analyses of alpha and beta diversity metrics (e.g. PERMANOVAs, NMDS, ANOVAs) and ANCOMBC2 analysis of differntial abundance of microbial taxa. For network analysis run "spieceasi_with_stats_forGithub.Rmd" and "succession-analyses-forGithub.Rmd". Fo analysis of total microbial DNA concentraions run "picogreen-analyses-forGithub.Rmd". Please note that paneled figures were joined in Abobe Illustrator, and some aesthetic-only changes were made to R-generated figures for clarity.

If desired, this script can be run without the previous steps by importing the "bacteria16S.trim.rds" and "ITS.trim.rds" objects, which contains the phyloseq object of preprocessed data. All additional necessary data files can be downloaded from the data folder in the repository.