microbiome-differential-abundance

name	microbiome-differential-abundance
description	Differential abundance testing for microbiome data using compositionally-aware methods like ALDEx2, ANCOM-BC2, and MaAsLin2. Use when identifying taxa that differ between experimental groups while accounting for the compositional nature of microbiome data.
tool_type	r
primary_tool	ALDEx2

Version Compatibility

Reference examples tested with: DESeq2 1.42+, ggplot2 3.5+, phyloseq 1.46+, scanpy 1.10+

Before using code patterns, verify installed versions match. If versions differ:

• R: packageVersion('<pkg>') then ?function_name to verify parameters

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

Differential Abundance Testing

"Find which taxa differ between my groups" -> Identify differentially abundant taxa between experimental conditions using compositionally-aware methods that account for the relative nature of microbiome data.

• R: ALDEx2::aldex() for CLR-transformed Welch's t-test
• R: ANCOMBC::ancombc2() for bias-corrected log-linear models
• R: Maaslin2::Maaslin2() for multivariable association

The Compositionality Problem

Microbiome data is compositional - abundances are relative, not absolute. Standard tests (t-test, DESeq2) can give false positives.

ALDEx2 (Recommended)

Goal: Identify differentially abundant taxa between groups using a compositionally-aware statistical framework.

Approach: Apply CLR transformation with Monte Carlo sampling on the OTU table, run Welch's t-test per taxon, and filter by FDR-corrected p-value and effect size.

library(ALDEx2)
library(phyloseq)

ps <- readRDS('phyloseq_object.rds')
otu <- as.data.frame(otu_table(ps))
if (!taxa_are_rows(ps)) otu <- t(otu)

# Define groups
groups <- sample_data(ps)$Group

# Run ALDEx2 (CLR transformation + Welch's t-test)
aldex_results <- aldex(otu, groups, mc.samples = 128, test = 'welch',
                       effect = TRUE, include.sample.summary = FALSE)

# Filter significant
sig_aldex <- aldex_results[aldex_results$we.eBH < 0.05 & abs(aldex_results$effect   


aldex.plotaldex_results type   test  

ANCOM-BC2 (Recommended)

library(ANCOMBC)

# Run ANCOM-BC2 with sensitivity analysis
ancom_result <- ancombc2(data = ps, fix_formula = 'Group',
                         p_adj_method = 'BH', pseudo_sens = TRUE,
                         prv_cut = 0.1, lib_cut = 1000,
                         group = 'Group', struc_zero = TRUE)

# Extract results (includes sensitivity analysis)
res_df <- ancom_result$res

# Primary results
sig_ancom <- res_df[res_df$diff_Group == TRUE, ]

# Check sensitivity (passed_ss = passed sensitivity analysis)
robust_hits <- res_df[res_df$diff_Group == TRUE & res_df$passed_ss_Group == TRUE, ]

MaAsLin2

library(Maaslin2)

# Prepare data
features <- as.data.frame(t(otu_table(ps)))
metadata <- as.data.frame(sample_data(ps))

# Run MaAsLin2
maaslin_results <- Maaslin2(
    input_data = features,
    input_metadata = metadata,
    output = 'maaslin2_output',
    fixed_effects = 'Group',
    normalization = 'CLR',
    transform = 'NONE',
    analysis_method = 'LM'
)

# Results in maaslin2_output/all_results.tsv
sig_maaslin <- maaslin_results$results[maaslin_results$results$qval < 0.05, ]

DESeq2 (with caution)

library(DESeq2)
library(phyloseq)

# Convert to DESeq2 (use geometric mean of poscounts)
ps_deseq <- ps
ps_deseq <- prune_samples(sample_sums(ps_deseq) > 1000, ps_deseq)

dds <- phyloseq_to_deseq2(ps_deseq, ~ Group)
dds <- DESeq(dds, test = 'Wald', fitType = 'parametric', sfType = 'poscounts')

res <- results(dds, alpha = 0.05)
sig_deseq <- res[which(res$padj < 0.05 & abs(res$log2FoldChange) > 1), ]

Visualization

library(ggplot2)

# Volcano plot from ALDEx2
ggplot(aldex_results, aes(x = effect, y = -log10(we.eBH))) +
    geom_point(aes(color = we.eBH < 0.05 & abs(effect) > 1), alpha = 0.6) +
    geom_hline(yintercept = -log10(0.05), linetype = 'dashed') +
    geom_vline(xintercept = c(-1, 1), linetype = 'dashed') +
    scale_color_manualvalues    
    theme_minimal 
    labsx   y  

Method Comparison

Method	Handles	Covariates	Speed	Notes
ALDEx2	Compositionality	Limited	Slow	Best for simple designs
ANCOM-BC2	Compositionality, zeros, sensitivity	Yes	Medium	Recommended for complex designs
MaAsLin2	Compositionality	Yes	Fast	Good for longitudinal
DESeq2	Sparsity (less ideal)	Yes	Fast	Use with caution for microbiome

Related Skills

• diversity-analysis - Identify overall differences first
• differential-expression/deseq2-basics - Similar concepts
• pathway-analysis/go-enrichment - Enrichment of differential taxa