Analysis bash scripts, processed data files, and R scripts for Functional plasticity in oyster gut microbiomes along a eutrophication gradient in an urbanized estuary

Study of wild oysters and their gut microbiomes along the estuarine gradient of Narragansett Bay, Rhode Island, USA with 16S rRNA amplicon and metatranscriptome data.

This repository contains the scripts, pre-processed sequencing data, and the R script files to reproduce the figures in the manuscript. The raw sequences generated for this study can be found in the NCBI Short Read Archive under BioProject no. PRJNA598635. The corresponding accession numbers for each sample are detailed in PRJNA598635_NCBI_info.xlsx. This file also includes file names and environmental data for each 16S rRNA amplicon or metatranscriptomic sample.

To cite this work:

Stevick, R.J., Post, A.F. & Gómez-Chiarri, M (2021). Functional plasticity in oyster gut microbiomes along a eutrophication gradient in an urbanized estuary. Animal Microbiome 3, 5. https://doi.org/10.1186/s42523-020-00066-0

This repository has been archived on Zenodo. Access or cite the most recent release:

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Contents

MetatranscriptomeAnalysis

This folder contains scripts used to perform modified SAMSA2 analysis on the metatranscriptomic data.

1. Bash Scripts

• Main modified SAMSA2 script (masterscript*.sh)
• Scripts to run Rscripts on the command line (deSEQ*.sh)
• Script to compute DIAMOND annotations against refSeq dbs (newannot.sh)
• Raw sequence counts and file inputs (rawcounts.txt and samples_transcriptomes.txt)

2. R_scripts

• DeSeq2 files to run states for the susbsytems based on deSEQ_groups.sh or deSEQ_subsys.sh

16SampliconAnalysis

This folder contains scripts used to perform QIIME2 analysis on the 16S rRNA amplicon data.

1. Scripts

• Main QIIME2 script with all commands (00_qiime2_alltogether.sh)
• Script to export QIIME2 files (qiime2_exportcollapsedtable.sh)

2. QIIME2 Results

• core-metrics-results: diversity metrics calculated by QIIME2
• exported: files exported from QIIME2, then imported to excel
• rarefaction: curves generated by QIIME2
• taxonomy: annotations and barplots generated by QIIME2
• all other qza and qzv files generated during the QIIME2 pipeline

TNCpaper_ScriptsData

This folder contains all the processed data files from the analysis described in MetatranscriptomeAnalysis & 16SampliconAnalysis, and the scripts to reproduce the figures and statistics in the manuscript. Each script is named for the figures it creates.

1. Function (from MetatranscriptomeAnalysis)

• DeSeq2 results for level-2 SEED functional annnotation of metatranscriptomes (each station vs. mean)
• DeSeq2 results for level-3 SEED functional annnotation of metatranscriptomes (each station vs. mean)
• DeSeq2 results for level-4 SEED functional annnotation of metatranscriptomes (each station vs. mean)
• DeSeq2 results for SEED functional annnotation of metatranscriptomes (North vs. South)

2. Metadata

• Folder containing mapping files to create Figure 1
• Metadata spreadsheet with oyster and environmental measurements

3. Taxonomy (from MetatranscriptomeAnalysis & 16SampliconAnalysis)

• 16S rRNA taxonomy results
• LefSe analysis results for the 16S rRNA taxa at the order level
• Metatranscriptomic annotation taxonomy results with RefSeq

Scripts

• Figure1_2_Table1.R - map of sites and PCA of environmental data, calculates environmental correlation coeffs for Table 1.
• Figure3_S1_S2_S6.R - 16S rRNA percent abundance barplots (including controls) (S1), rarefaction plots (S2), and alpha- and beta-diversity (3). 16S core microbiome (S6). Table S2 of beta-diversity stats.
• Figure4_S2_S3_S5_S6.R - compares taxonomy generated from RefSeq/metatranscriptomes and 16S rRNA data with heatmaps and UpSetR plots (4 and S3). Metatranscriptome taxonomy rarefaction (S2) and beta-diversity plots (S5). Metatranscriptome core microbiome (S6).
• Figure5_6.R - Metaranscriptomic functional data at pathway levels, subset for stress response (N vs S), nitrogen and phosphorus metabolism (each site vs mean) with barplots.
• FigureS4_lefse.R - 16S rRNA amplicon data at order level output from LefSe (Taxonomy/TNC_summaryLefSeResults.xlsx)
• FigureS7-S10.R - Metaranscriptomic functional data at gene level for stress response, nitrogen metabolism, phosphorus metabolism (heatmaps)