Microbiome Analysis Toolkit (R/phyloseq-based)

This repository provides a comprehensive R-based toolkit for microbiome data analysis using the phyloseq package. It covers alpha/beta diversity, taxonomic analysis, statistical testing, and publication-ready visualization including volcano plots and bar plots. Below is a structured README describing each function module and usage example.

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Requirements

R packages

install.packages(c("phyloseq", "ggplot2", "dplyr", "ggtext", "ggprism", "ggrepel", "patchwork", "broom", "vegan", "glue", "ggpubr", "tidyr", "purrr", "rvg", "officer", "combinat", "RColorBrewer", "GGally"))

source("./250429_microbiome_analysis_functions.R")

1. Alpha Diversity

Function: create_alpha_diversity_df()

• Calculates richness metrics: Observed, Shannon, Chao1, Inverse Simpson.
• Optional: Faith's PD (requires tree).

alpha_df <- create_alpha_diversity_df(physeq_obj, tree = TRUE)

Function: alpha_plot()

• Plots violin + boxplot + significance tests (Kruskal, Wilcoxon).

alpha_plot(alpha_df, x_ = "Group", y_ = "Shannon")

Function: alpha_df()

• Outputs CI & p-values for each index using Kruskal-Wallis and Wilcoxon.

res <- alpha_df(df = alpha_df, group = "Group")

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2. Beta Diversity

Function: beta_plot()

• Computes PERMANOVA, ANOSIM, PERMDISP.
• Visualizes PCoA with ellipse.

beta_out <- beta_plot(phyloseq, type = "Group", type_col = c("red", "blue"))

Function: beta_plot.envfit()

• Extension of beta_plot() with vegan::envfit() support and global PERMANOVA.

beta_env <- beta_plot.envfit(phyloseq, type = "Group", envfit = TRUE, formula = "Age + BMI", PERMANOVA = TRUE)

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3. Taxonomic Abundance & Visualization

Function: Abund_cal()

• Computes mean, sd, se, CI per taxon and group.

Abund_cal(ps.glom, tax_level = "Genus", group = "Group", path = "./results/")

Function: taxa_plot() / tax_plot.sp()

• Visualizes top taxa at any level or within a genus.
• Automatically color-coded by phylum.

tax_plot_out <- taxa_plot(melt, taxa = "Genus", tax_otu = otu_list, x_axis = "SampleID")

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4. Mean Abundance Comparison (with Statistics)

Function: mean_plot() / mean_plot.sp()

• Generates comparative plots: bar, log2FC, p-value, heatmap.
• Returns patchwork plot and statistics.

res <- mean_plot(melt, tax_level = "Genus", tax_otu = top_genera, type = "Group", palet = c("#E31A1C", "#1F78B4"))

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5. Volcano Plot

Function: volcano_plot()

• Computes Wilcoxon p-values, log2FC, and highlights significant taxa.

vol_out <- volcano_plot(ph_glom, tax_rank = "Species", comparision = "Group", compar1 = "A", compar2 = "B")

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6. BLAST Annotation Processing

Function: blast_arrange()

• Extracts Genus, Species names from BLAST sscinames output.

blast_table <- blast_arrange(blast_output, ASV = "Query")

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7. Prevalence & Abundance Filtering

Function: filter_phyloseq_by_prevalence_abundance()

• Filters OTUs based on prevalence and average abundance thresholds.

ps_filtered <- filter_phyloseq_by_prevalence_abundance(ps, 0.001, 0.1)

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8. Miscellaneous Utilities

• save_ggplot_to_pptx(): Saves ggplot as editable PowerPoint.
• ps_tax_update(): Updates ASV ID based on abundance and reassigns taxonomy table.
• seq_to_fas(): Converts sequence list to FASTA.

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Author

This script was developed by Soyeon Kim for microbiome analysis pipelines based on the phyloseq R package.
It is part of a broader effort to support microbial ecology, statistical testing, and reproducible bioinformatics workflows.

📧 Contact: kim.soyeon.bio@gmail.com
🔗 Related blog post: https://bio-kcs.tistory.com/

License

This repository is licensed under the MIT License.
You are free to use, modify, and distribute the code with attribution.