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Content from Introduction to metagenomics

Last updated on 2026-04-28 | Edit this page

Overview

Questions

  • What is metagenomics?
  • What are the applications of metagenomics?
  • What are the advantages of using metagenomics?
  • What is the difference between metagenomic profiling and genome-resolved metagenomics (MAGs)

Objectives

  • Define metagenomics.
  • Explain the difference between metagenomics and amplicon sequencing.
  • Describe main approaches for metagenomic profiling.
  • Explain what a “metagenome-assembled genome” (MAG) is.
  • Bonus: be able to explain that to a layperson at a party!
Key Points
  • Metagenomics sequences all DNA, giving an unbiased, high-resolution view of community composition and function—beyond what amplicon sequencing can capture.
  • Reads profiling delivers fast, reference-based taxonomic and functional counts data for downstream analyses like TaxSEA and integration workflows.
  • MAGs can be reconstructed via assembly and binning of reads, revealing uncultured lineages and their metabolic potential.
  • Modern sequencing and workflows (short-reads, long-reads, hybrids; nf-core pipelines) improve accuracy, assembly quality, and reproducibility.

Content from Taxon-set enrichment analysis with TaxSEA

Last updated on 2026-04-28 | Edit this page

Overview

Questions

  • How can we test whether groups of related taxa (taxon sets) show coordinated shifts between conditions?
  • What input does TaxSEA need, and how should taxonomic IDs be formatted?
  • How does TaxSEA extend differential abundance results using enrichment analysis?
  • How can we interpret enriched taxon sets to understand functional, ecological, or disease-associated patterns?

Objectives

  • Prepare species- or genus-level differential abundance results for use with TaxSEA.
  • Run TaxSEA to test for enrichment across predefined or custom taxon sets.
  • Understand the structure of TaxSEA output, including P values, effect direction, and FDR.
  • Interpret enrichment results in the context of microbiome function, ecology, and host associations.

Authors: Calum J. Walsh & Feargal J. Ryan

TaxSEA is a Bioconductor package that helps microbiome researchers test for enrichment in known microbial signatures, including:

  • Metabolite producers
  • Disease associations
  • Previously published microbiome signatures
  • Traits established from in-vitro data

TaxSEA takes as input a vector of genus or species names and a rank (e.g. log2 fold changes or Spearman’s rho) and uses a Kolmogorov-Smirnov test to identify if a particular group of species or genera (i.e. a set of taxa such as butyrate producers) are skewed to one end of the distribution (i.e. enriched).

TaxSEA is based on the concept of Gene Set Enrichment Analysis (GSEA). For an overview, check out the link here:https://www.metwarebio.com/gsea-enrichment-analysis-guide/.

Note: Although TaxSEA can, in principle, be applied to microbiome data from any source, the databases utilised largely cover human-associated microbiomes, particularly the human gut microbiome. As such TaxSEA will likely perform best on human gut microbiome data.

Taxon set database

By default TaxSEA utilises taxon sets generated from reference databases (gutMGene, GMrepo v2, MiMeDB, mBodyMap, BugSigDB, BacDive) as well as a handful of sets curated from the literature (mucin utilisers and BLOSSUM taxa). See below for examples of using custom databases or taxonomically-defined taxon sets.

Please cite the appropriate database if using:

  • Cheng et al. gutMGene: a comprehensive database for target genes of gut microbes and microbial metabolites Nucleic Acids Res. 2022.
  • Dai et al. GMrepo v2: a curated human gut microbiome database with special focus on disease markers and cross-dataset comparison Nucleic Acids Res. 2022.
  • Wishart et. al. MiMeDB: the Human Microbial Metabolome Database Nucleic Acids Res. 2023.
  • Jin et al. mBodyMap: a curated database for microbes across human body and their associations with health and diseases. Nucleic Acids Res. 2022.
  • Geistlinger et al. BugSigDB captures patterns of differential abundance across a broad range of host-associated microbial signatures. Nature Biotech. 2023.

Load the Package

R 

library(TaxSEA)

Input

All that is required for TaxSEA is an R vector containing ranks (e.g. log2 fold changes) and names (e.g. species/genus). TaxSEA will not work if input names are from ranks higher than species or genus and will struggle with GTDB taxonomy. The input should include all taxa tested, not a limited or pre-defined set (e.g. do not use a threshold for significance or remove any taxa). TaxSEA will lookup and convert taxon names to NCBI taxonomic identifiers. TaxSEA stores a commonly observed identifiers internally and so will only lookup whatever is not covered to save time.

Input IDs should be formatted like one of the following:

  • Species name. E.g. “Bifidobacterium longum”, “Bifidobacterium_longum”
  • Genus name. E.g. “Bifidobacterium”
  • NCBI ID E.g. “216816”

The code chunk below is useful for splitting names that contain the full species taxonomy (eg. MetaPhlAn output). We don’t need to run it now.

R 

# Input IDs with the full taxonomic lineage should be split E.g.
x <- "d__Bacteria.p__Actinobacteriota.c__Actinomycetes.o__Bifidobacteriales.f__Bifidobacteriaceae.g__Bifidobacterium"
x <- strsplit(x, split = "\\.")[[1]][6]
x <- gsub("g__", "", x)


# Running this through a vector of IDs may look something like the following
new_ids <- sapply(as.character(old_ids), function(y) {strsplit(x = y, split = "\\.")[[1]][6]})
new_ids <- gsub("g__", "", new_ids)

Load and Inspect Test Data

The TaxSEA library comes with pre-processed test data included.
The data comes from a study comparing IBD patients to healthy controls.
The count data was downloaded from curatedMetagenomicData and fold changes between the two groups were generated with LinDA.

We can load this data into our working environment using the data() function

R 

data("TaxSEA_test_data")

sample(TaxSEA_test_data, 4)

OUTPUT 

 Haemophilus_sp_HMSC71H05          Dorea_sp_CAG_317 Monoglobus_pectinilyticus
                    2.179                     2.162                    -1.069
     Actinomyces_sp_ICM47
                    1.539

The input is a vector of species names and log fold changes. Perfect for TaxSEA.

Run TaxSEA with Test Data

R 

taxsea_results <- TaxSEA(taxon_ranks = TaxSEA_test_data)

Examine outputs

Output

The output is a list of data frames providing enrichment results for metabolite produers, health/disease associations, and published signatures from BugSigDB. Each dataframe has at least 6 columns:

  1. taxonSetName - The name of the taxon set tested
  2. median_rank_of_set_members - This is simply the median rank across all detected members in the set. This allows you to see the direction of change
  3. PValue - Kolmogorov-Smirnov test P value.
  4. Test_statistic - Kolmogorov-Smirnov test statistic.
  5. FDR - P value adjusted for multiple testing.
  6. TaxonSet - Returns list of taxa in the set to show what is driving the signal

All results: This includes all databases. Is useful for a quick look and overview but can get a bit messy.

R 

all_results.df <- taxsea_results$All_databases

Metabolite Producers: Enrichment among metabolite producers from gutMgene and MiMeDB

R 

metabolites.df <- taxsea_results$Metabolite_producers

Health Associations: Enrichment among health and disease signatures from GMRepoV2 and mBodyMap

R 

disease.df <- taxsea_results$Health_associations

BugSigDB: Enrichment among published associations from BugSigDB - a database of manually curated microbial signatures from published differential abundance studies. The output format here is a bit different. You’ll notice that, where available, it includes a PubMed ID linking back to the reporting paper.

R 

bsdb.df <- taxsea_results$BugSigDB

Bacterial Physiology: Enrichment among bacterial meta data from from BacDive - a standardised database of bacterial information, including physiology.

R 

bacdive.df <- taxsea_results$BacDive_bacterial_physiology

Visualisation of TaxSEA output

As TaxSEA operates on individual species and ranks, the easiest way to visualise these is often through the input ranks. For example using volcano plots, or density plots can be intuitive and useful ways to see the overall trend in a set and the members driving it. Remember just becomes an individual species isn’t significant by itself, doesn’t mean that the set cannot be significant.

R 

library(ggplot2)
library(gridExtra)
library(reshape)
library(curatedMetagenomicData)
library(TaxSEA)
library(MicrobiomeStat)
library(ggrepel)
library(ggpubr)
library(ggridges)

########################
#### HallAB_2017 ####
########################
allmeta_data = sampleMetadata

# HallAB_2017 dataset
HallAB_2017_cmd_object = curatedMetagenomicData(pattern = "2021-10-14.HallAB_2017.relative_abundance",counts = TRUE,dryrun = FALSE)

R 

HallAB_2017_counts.df = HallAB_2017_cmd_object
HallAB_2017_counts.df = (HallAB_2017_counts.df$`2021-10-14.HallAB_2017.relative_abundance`)

HallAB_2017_counts.df = assay(HallAB_2017_counts.df)
HallAB_2017_counts.df = HallAB_2017_counts.df[apply(HallAB_2017_counts.df>1000,1,sum)>4,]
HallAB_2017_md.df = allmeta_data[allmeta_data$sample_id %in% colnames(HallAB_2017_counts.df),]
rownames(HallAB_2017_md.df) = HallAB_2017_md.df$sample_id

## Ensure data is matching
colnames(HallAB_2017_counts.df) %in% rownames(HallAB_2017_md.df)

OUTPUT 

  [1] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
 [16] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
 [31] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
 [46] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
 [61] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
 [76] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
 [91] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
[106] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
[121] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
[136] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
[151] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
[166] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
[181] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
[196] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
[211] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
[226] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
[241] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
[256] TRUE TRUE TRUE TRUE

R 

HallAB_2017_counts.df = HallAB_2017_counts.df[,rownames(HallAB_2017_md.df)]
HallAB_2017_md.df = HallAB_2017_md.df[,c("study_name","sample_id","subject_id","study_condition","age_category","DNA_extraction_kit","visit_number")]

## Remove duplicate samples 
HallAB_2017_md.df = HallAB_2017_md.df[order(HallAB_2017_md.df$visit_number,decreasing = FALSE),]
HallAB_2017_md.df = HallAB_2017_md.df[!duplicated(HallAB_2017_md.df$subject_id),]
HallAB_2017_counts.df = HallAB_2017_counts.df[,rownames(HallAB_2017_md.df)]

## Assigning rows as species names ##
spec_vec = sapply(as.character(rownames(HallAB_2017_counts.df)),function(y) {strsplit(x = y,split="\\|")[[1]][7]})
names(spec_vec) = NULL
spec_vec = gsub("s__","",spec_vec)
rownames(HallAB_2017_counts.df) = spec_vec

HallAB_2017_linda_res = linda(HallAB_2017_counts.df, 
                              HallAB_2017_md.df, 
                              formula = '~study_condition+DNA_extraction_kit')

OUTPUT 

0  features are filtered!
The filtered data has  32  samples and  299  features will be tested!

OUTPUT 

Imputation approach is used.
Fit linear models ...
Completed.

R 

HallAB_2017_ranks = HallAB_2017_linda_res$output$study_conditionIBD$log2FoldChange
names(HallAB_2017_ranks) = rownames(HallAB_2017_linda_res$output$study_conditionIBD)
HallAB_2017_TaxSEA_results.df = TaxSEA(taxon_ranks = HallAB_2017_ranks)

alldb = HallAB_2017_TaxSEA_results.df$All_databases  
HallAB_2017_DA = HallAB_2017_linda_res$output$study_conditionIBD

hall_bacterialids = get_ncbi_taxon_ids(rownames(HallAB_2017_DA))
data("TaxSEA_db")

scfas = unique(unlist(c(TaxSEA_db$GutMGene_producers_of_Propionate,
                        TaxSEA_db$GutMGene_producers_of_Butyrate)))

HallAB_2017_DA$NCBI_id = hall_bacterialids[rownames(HallAB_2017_DA)]
HallAB_2017_DA$Nitrate_utilisers = HallAB_2017_DA$NCBI_id %in% TaxSEA_db$BacDive_Utilizes_nitrate
HallAB_2017_DA$Faculatative_anerobes = HallAB_2017_DA$NCBI_id %in% TaxSEA_db$`BacDive_facultative anaerobe`
HallAB_2017_DA$SCFA_producers = HallAB_2017_DA$NCBI_id %in% scfas
HallAB_2017_DA$Species = rownames(HallAB_2017_DA)
HallAB_2017_DA$mBodyMap_skin = HallAB_2017_DA$NCBI_id %in% TaxSEA_db$mBodyMap_skin


volcano_plot_all = ggplot(HallAB_2017_DA,aes(x=log2FoldChange,y=-log10(padj)))+
  geom_point(color="grey33",size=1)+
  theme_classic()+guides(color="none",size="none")+
  geom_label_repel(data=HallAB_2017_DA[HallAB_2017_DA$padj < 0.1,],
                   aes(label=Species))+
  geom_vline(xintercept = 0,linetype=3)+
  geom_hline(yintercept = -log10(0.1),linetype=3)+
  ggtitle("All Species")



scfa_volcano = ggplot(HallAB_2017_DA,aes(x=log2FoldChange,y=-log10(padj),
                               colour=SCFA_producers,
                               size=SCFA_producers))+
  geom_point()+
  scale_colour_manual(values=c("grey33","deepskyblue3"))+
  scale_size_manual(values=c(1,3))+
  theme_classic()+guides(color="none",size="none")+
  geom_label_repel(data=HallAB_2017_DA[HallAB_2017_DA$SCFA_producers==TRUE,],
                   aes(label=Species),max.overlaps = 4)+
  ggtitle("SCFA producers")



volcano_fa = ggplot(HallAB_2017_DA,aes(x=log2FoldChange,y=-log10(padj),
                               colour=Faculatative_anerobes,
                               size=Faculatative_anerobes))+
  geom_point()+
  scale_colour_manual(values=c("grey33","darkorchid"))+
  scale_size_manual(values=c(1,3))+
  theme_classic()+guides(color="none",size="none")+
  geom_label_repel(data=HallAB_2017_DA[HallAB_2017_DA$Faculatative_anerobes==TRUE,],
                   aes(label=Species))+
  ggtitle("Faculatative anerobes")

ggarrange(volcano_plot_all,volcano_fa,scfa_volcano,ncol = 3,nrow=1)

R 

 ## Build a long df for the ridge plot
 ridge_dat <- rbind(
   data.frame(log2FoldChange = HallAB_2017_DA$log2FoldChange,
              Group = "All taxa"),
   data.frame(log2FoldChange = HallAB_2017_DA$log2FoldChange[HallAB_2017_DA$SCFA_producers],
              Group = "SCFA producers"),
   data.frame(log2FoldChange = HallAB_2017_DA$log2FoldChange[HallAB_2017_DA$mBodyMap_skin],
              Group = "Skin-associated"),
   data.frame(log2FoldChange = HallAB_2017_DA$log2FoldChange[HallAB_2017_DA$Faculatative_anerobes],
              Group = "Facultative anaerobes")
 )
 
 ridge_dat$Group <- factor(
   ridge_dat$Group,
   levels = rev(c("All taxa",
                  "SCFA producers",
                  "Skin-associated",
                  "Facultative anaerobes"))
 )
 
 ggplot(ridge_dat,
        aes(x = log2FoldChange,
            y = Group,
            fill = Group)) +
   geom_density_ridges(alpha = 0.85, scale = 1.2, colour = NA) +
   xlim(-10, 10) +
   geom_vline(xintercept = 0, linetype = 3) +
   theme_classic() +
   scale_fill_manual(values = rev(c(
     "grey44",        # All taxa
     "deepskyblue3",  # SCFA
     "tan3",          # Skin
     "darkorchid"     # Anaerobes
   ))) +
   theme(legend.position = "none") +
   ylab(NULL) +
   ggtitle("log2FC distributions for functional/ecological groups")

Custom databases

If you want to run TaxSEA with your own database, use the custom_db argument. It should be a named list where:

  • each name is the label for a set, and
  • each value is a vector of species assigned to that set.

This is the same format as the default TaxSEA database.

Note: using the custom_db flag disables the automatic ID conversion and NCBI API lookup. However this is available via other functions.

Perform enrichment analysis using TaxSEA with custom databases

In addition to taxon sets defined by function or phenotype, users can define custom sets based on taxonomy. Current methods to test differential abundance at higher taxonomic levels (e.g., genus or family) involve aggregating counts, but with this approach opposing shifts in individual species may cancel each other out, obscuring meaningful biological patterns. For instance, antibiotic treatment may suppress certain species while allowing resistant species within the same genus to expand and occupy the vacant niche, creating an ecological shift that appears as no net change at broader taxonomic levels. Here we utilise data from Chng et al. 2016, a study comparing the skin microbiome between Atopic dermatitis and controls.

The script below applies the TaxSEA framework to identify taxonomic enrichment at different taxonomic levels. Specifically, we analyse enrichment at the order level from metagenomic data.

The code block below does the following:

  • Downloads species abundance data
  • Performs some light QC
  • Defines taxonomic sets at each level of taxonomy (phylum -> genus)
  • Calculates differential abundance between groups
  • Performs enrichment analysis using TaxSEA
  • Plots results

R 

# Load required libraries
library(TaxSEA)
library(curatedMetagenomicData)
library(tidyverse)
library(phyloseq)
library(MicrobiomeStat)
library(dplyr)
library(DT)

# Load sample metadata from curatedMetagenomicData
metadata_all <- sampleMetadata

# Filter metadata for the specific study (ChngKR_2016)
metadata <- metadata_all %>% 
  filter(study_name == "ChngKR_2016") %>% 
  column_to_rownames('sample_id')

# Extract count data from curatedMetagenomicData 
cmd_data <- curatedMetagenomicData(
  pattern = "ChngKR_2016.relative_abundance",
  counts = TRUE,
  dryrun = FALSE
)

R 

# Convert the extracted data to a count matrix
counts_data <- assay(cmd_data[[1]])
# Subset to relevant samples
counts_data <- counts_data[, rownames(metadata)]  

# Filter taxa to only keep those with at least one sample having counts > 100
counts_data <- counts_data[apply(counts_data > 100, 1, sum) > 0, ]

# Extract species names from taxonomic strings
species_names <- gsub("s__", "", sapply(rownames(counts_data), function(y) strsplit(y, "\\|")[[1]][7]))
rownames(counts_data) <- species_names

# Create a taxonomic lineage data frame to define Sets
# Remove taxonomic prefixes (k__, p__, c__, etc.) and separate into taxonomic ranks
taxon_lineages <- data.frame(Name = species_names,
                             Lineage = names(species_names)) %>%
  mutate(Lineage = str_remove_all(Lineage, '[kpcofgs]__')) %>%
  separate(col = Lineage, into = c('kingdom', 'phylum', 'class', 
                                   'order', 'family', 'genus', 'species'), 
           sep = '\\|') %>%
  mutate(name = Name) %>%
  remove_rownames() %>%
  column_to_rownames('name')

# Perform differential abundance testing using LinDA
metadata$study_condition <- factor(metadata$study_condition, levels = c("control", "AD"))

linda_results <- linda(
  feature.dat = counts_data,
  meta.dat = metadata,
  formula = '~study_condition',
  feature.dat.type = 'count',
  prev.filter = 0.05
)

OUTPUT 

216  features are filtered!
The filtered data has  78  samples and  216  features will be tested!
Pseudo-count approach is used.
Fit linear models ...
Completed.

R 

# Extract log2 fold change values for differential taxa
linda_results <- linda_results$output$study_conditionAD
log2_fold_changes <- linda_results$log2FoldChange
names(log2_fold_changes) <- rownames(linda_results)


# Create a named list of species grouped by order
custom_taxon_sets <- taxon_lineages %>%
  group_by(.data[['order']]) %>% 
  summarise(species = list(species), .groups = "drop") %>%
  deframe()

# Perform enrichment analysis using TaxSEA
custom_taxsea_results <- TaxSEA(taxon_ranks = log2_fold_changes, custom_db = custom_taxon_sets)
custom_taxsea_results <- custom_taxsea_results$custom_sets

datatable(custom_taxsea_results)

R 

# plotting
data.frame(Name = names(log2_fold_changes),
           Log2FC = log2_fold_changes) %>%
  left_join(taxon_lineages) %>%
  left_join(custom_taxsea_results, by = c("order" = "taxonSetName")) %>%
  mutate(Direction = if_else(FDR > 0.1, 'Insig.', if_else(median_rank_of_set_members > 0, 'Up', 'Down'))) %>%
  drop_na(PValue) %>%
  ggplot(aes(x = Log2FC, y = order, group = order)) +
  geom_density_ridges(aes(fill = Direction)) +
  theme_bw() +
  scale_fill_manual(values = c('Insig.' = 'grey80',
                              'Up' = 'steelblue',
                              'Down' = 'firebrick'))
Key Points
  • TaxSEA performs taxon-set enrichment analysis on differential abundance results, similar to GSEA.
  • Inputs must be species or genus names with corresponding ranks (e.g., log2 fold changes).
  • TaxSEA tests whether members of a taxon set are skewed toward one end of the ranked distribution.
  • Enrichment results help reveal functional or ecological patterns that may not be visible at the single-taxon level.

Content from Multi-omics integration with DIABLO

Last updated on 2026-04-28 | Edit this page

Overview

Questions

  • How can we integrate microbiome and metabolomics data to identify shared biological signals?
  • What does DIABLO do, and how is it different from single-omics analyses?
  • How do we tune a multi-omics model and choose the number of components and selected features?
  • How do we interpret DIABLO outputs to understand discriminative multi-omics signatures?

Objectives

  • Preprocess microbiome and metabolomics data for multi-omics integration (filtering, transformation, normalisation).
  • Perform unsupervised integration (sPLS) to explore cross-omics correlations.
  • Train, tune, and validate a DIABLO model for classification.
  • Interpret sample plots, feature correlations, loadings, and multi-omics signatures produced by DIABLO.

Author: Geraldine Kong

This workshop introduces multi-omics integration using DIABLO from mixOmics R package, adapted from mixOmics’ tutorial https://mixomics.org/mixdiablo/diablo-tcga-case-study/.

We will use the dataset from Franzosa et al. (2019) to integrate microbiome species profiles from shotgun metagenomics with metabolomics data. The goal is to identify a multi-omics signature that discriminates healthy controls from patients with Clostridium difficile (CD) by finding correlated and predictive features across the omics. You will learn how to tune the model, explore the relationships between features, and interpret sample groupings.

Setting up

First, load the required packages and the helper functions for data pre-processing.

R 

library(mixOmics)

OUTPUT 

Loading required package: MASS

OUTPUT 

Loading required package: lattice

OUTPUT 

Loading required package: ggplot2

OUTPUT 


Loaded mixOmics 6.34.0
Thank you for using mixOmics!
Tutorials: http://mixomics.org
Bookdown vignette: https://mixomicsteam.github.io/Bookdown
Questions, issues: Follow the prompts at http://mixomics.org/contact-us
Cite us:  citation('mixOmics')

R 

# Prevalence filter function
## Usage example: filtered <- filter_prevalence(count_mat, min_prev = 0.20) # To filter features present in 20% of sample
## Samples as rows, Features as columns
filter_prev <- function(x, min_prev) {
  x <- as.matrix(x)
  if (!is.numeric(x)) {
    stop("Input data must be numeric (samples x features).")
  }
  
  # Calculate prevalence (number of non-zero values) per feature
  prev <- colSums(x > 0)
  n_samples <- nrow(x)
  
  # Convert proportion threshold to number of samples if needed
  if (min_prev < 1) {
    cutoff <- ceiling(min_prev * n_samples)
  } else {
    cutoff <- min_prev
  }
  
  # Keep only features meeting prevalence requirement
  keep <- prev >= cutoff
  
  # Display message
  message("Kept ", sum(keep), " / ", ncol(x), 
          " features (", round(sum(keep) / ncol(x) * 100, 2), "%) ",
          "that are present in ≥ ", min_prev * 100, "% of all samples.")
  
  return(x[, keep, drop = FALSE])
  
}

# Abundance filter function
### threshold: proportion cutoff (0.001 = 0.1%)
### Samples as rows, Features as columns
filter_microb_abund <- function(x, threshold = 0.001) {
  x <- as.matrix(x)
  if (!is.numeric(x)) {
    stop("Input data must be numeric (samples x features).")
  }
  
  # total count across all samples
  total_counts <- sum(x)
  
  # total abundance per feature (column sums)
  feature_totals <- colSums(x)
  
  # proportion of total for each feature
  feature_prop <- feature_totals / total_counts
  
  # keep features ≥ threshold
  keep <- feature_prop >= threshold
  
  # Display message
  message("Kept ", sum(keep), " / ", ncol(x), 
          " features (", round(sum(keep) / ncol(x) * 100, 2), "%) ",
          "with ≥ ", threshold * 100, "% total abundance")
  
  return(x[, keep, drop = FALSE])
}


# rCLR normalization function
### Samples as rows, Features as columns
rclr_norm <- function(x, pseudocount = 1, base = exp(1)) {
  x <- as.matrix(x)
  if (!is.numeric(x)) {
    stop("Input data must be numeric (samples x features).")
  }
  
  x <- x + pseudocount # add pseudocount to avoid log(0)
  
  lx <- log(x, base = base) # log-transform
  
  # robust center per sample (median of log-values)
  centers <- matrixStats::rowMedians(lx)
  
  # subtract median from each sample
  lx_centered <- sweep(lx, 1, centers, FUN = "-")
  
  return(lx_centered)
}

# Filter and transformation function for metabolomics data
### Samples as rows, features as columns
filter_metab_abund <- function(x, cutoff = 1000, min_prop = 0.7) {
  # Data: samples x features numeric matrix / data.frame
  # cutoff: intensity threshold

  if (!is.numeric(as.matrix(x))) {
    stop("Input x must be numeric (samples x features).")
  }
  
  keep <- apply(x, 2, function(x) mean(x > cutoff, na.rm = TRUE) >= min_prop)
  filtered <- x[, keep, drop = FALSE]
  
  attr(filtered, "removed_features") <- colnames(x)[!keep]
  attr(filtered, "kept_features") <- colnames(filtered)
  
  # Display message
  message("Kept ", sum(keep), " / ", ncol(x), 
          " features (", round(sum(keep) / ncol(x) * 100, 2), "%) ",
          "that passed the threshold in ≥ ", min_prop * 100, "% of samples.")
  
  return(filtered)
}

# Transform metabolomics data
transform_metab <- function(x, pseudocount    = 1e-6, # constant added constant to avoid log(0)
                            center = FALSE, scale  = FALSE) {
  # metab: samples x features numeric matrix / data.frame
  
  # coerce to matrix and check
  x <- as.matrix(x)
  if (!is.numeric(x)) {
    stop("Input 'metab' must be numeric (samples x features).")
  }
  
  rn <- rownames(x)
  cn <- colnames(x)
  
  ## 1. Sample-wise median normalization (PQN-like)
  metab_norm <- t(apply(x, 1, function(x) x / stats::median(x, na.rm = TRUE)))
  
  ## 2. Log10 transform
  metab_log <- log10(metab_norm + pseudocount)
  
  ## 3. Autoscaling (mean-center + unit variance by default)
  metab_scaled <- scale(metab_log, center = center, scale = scale)
  
  # restore dimnames
  rownames(metab_scaled) <- rn
  colnames(metab_scaled) <- cn
  
  return(metab_scaled)
}

Next, load the Franzosa2019 dataset and inspect the R objects. The data has 144 samples: 56 Controls and 88 CD cases, stored in an R list object containing the following:

  • metadata: 144 rows (samples) with 7 columns (sample metadata)

  • microbiome: 144 rows (samples) with 55882 columns (bacterial species)

  • metabolome: 144 rows (samples) with 367 columns (metabolites)

R 

data_int <- readRDS('data/Franzosa_IBD_2019.RDS')

# Inspect
lapply(data_int, dim)

OUTPUT 

$metadata
[1] 144   7

$microbiome
[1]   144 55882

$metabolome
[1] 144 367

R 

## You can view each object in Rstudio using View(data_int$metadata)

# Save to each object name
meta <- data_int$metadata
metab <- data_int$metabolome
microb <- data_int$microbiome

# Number of samples in each group
table(meta$Group)

OUTPUT 


     CD Control
     88      56

R 

# Check that the samples are in the same order across all datasets
table(rownames(microb) == meta$Sample)

OUTPUT 


TRUE
 144

R 

table(rownames(metab) == meta$Sample)

OUTPUT 


TRUE
 144

Microbiome data pre-processing

Filter the data

Features that are rare or with low abundance should be filtered out prior to analysis to remove noise from the data. Here we are filtering out microbiome features that are NOT present in more than 50% of the samples, AND have less than 0.1% overall abundance.

R 

# Filter based on prevalence 
## Drop rare features that are not present in more than 50% of the samples
microb_filt <- filter_prev(microb, min_prev = 0.5) # 50% of samples, or 70%

OUTPUT 

Kept 5664 / 55882 features (10.14%) that are present in ≥ 50% of all samples.

R 

# Filter based on relative abundance
## Drop features that have total of < 0.1%
microb_filt_0.1 <- filter_microb_abund(microb_filt, threshold = 0.001)

OUTPUT 

Kept 188 / 5664 features (3.32%) with ≥ 0.1% total abundance

Transform the data

Data should be normalised and transformed prior to integration. Here we apply the robust centered log-ratio transformation (rCLR) method for microbiome data which is less sensitive to extreme values than the standard CLR. A small pseudocount is added prior to rCLR transformation to avoid log(0).

R 

# rCLR transform data
microb_rclr <- rclr_norm(microb_filt_0.1, pseudocount = 1)
dim(microb_rclr)

OUTPUT 

[1] 144 188

Cleanup names

Because the original microbiome features contain the full taxonomic lineage (very long names), the code below shortens them to species names only.

R 

head(colnames(microb_rclr))

OUTPUT 

[1] "d__Bacteria;p__Firmicutes_A;c__Clostridia;o__Lachnospirales;f__Lachnospiraceae;g__Fusicatenibacter;s__Fusicatenibacter saccharivorans"
[2] "d__Bacteria;p__Verrucomicrobiota;c__Verrucomicrobiae;o__Verrucomicrobiales;f__Akkermansiaceae;g__Akkermansia;s__Akkermansia muciniphila_A"
[3] "d__Bacteria;p__Bacteroidota;c__Bacteroidia;o__Bacteroidales;f__Tannerellaceae;g__Parabacteroides;s__Parabacteroides goldsteinii"
[4] "d__Bacteria;p__Firmicutes_A;c__Clostridia;o__Lachnospirales;f__Lachnospiraceae;g__Blautia_A;s__Blautia_A faecis"
[5] "d__Bacteria;p__Firmicutes_A;c__Clostridia;o__Lachnospirales;f__Lachnospiraceae;g__Anaerostipes;s__Anaerostipes hadrus_A"
[6] "d__Bacteria;p__Firmicutes_A;c__Clostridia;o__Oscillospirales;f__Ruminococcaceae;g__Gemmiger;s__Gemmiger qucibialis"

R 

# Shorten species names
colnames(microb_rclr) <- sapply(colnames(microb_rclr), function(x){
  strsplit(x, split = ';')[[1]][7]
})
colnames(microb_rclr) <- gsub("s__", "", colnames(microb_rclr))

# New names
head(colnames(microb_rclr))

OUTPUT 

    d__Bacteria;p__Firmicutes_A;c__Clostridia;o__Lachnospirales;f__Lachnospiraceae;g__Fusicatenibacter;s__Fusicatenibacter saccharivorans
                                                                                                        "Fusicatenibacter saccharivorans"
d__Bacteria;p__Verrucomicrobiota;c__Verrucomicrobiae;o__Verrucomicrobiales;f__Akkermansiaceae;g__Akkermansia;s__Akkermansia muciniphila_A
                                                                                                              "Akkermansia muciniphila_A"
          d__Bacteria;p__Bacteroidota;c__Bacteroidia;o__Bacteroidales;f__Tannerellaceae;g__Parabacteroides;s__Parabacteroides goldsteinii
                                                                                                            "Parabacteroides goldsteinii"
                          d__Bacteria;p__Firmicutes_A;c__Clostridia;o__Lachnospirales;f__Lachnospiraceae;g__Blautia_A;s__Blautia_A faecis
                                                                                                                       "Blautia_A faecis"
                  d__Bacteria;p__Firmicutes_A;c__Clostridia;o__Lachnospirales;f__Lachnospiraceae;g__Anaerostipes;s__Anaerostipes hadrus_A
                                                                                                                  "Anaerostipes hadrus_A"
                       d__Bacteria;p__Firmicutes_A;c__Clostridia;o__Oscillospirales;f__Ruminococcaceae;g__Gemmiger;s__Gemmiger qucibialis
                                                                                                                    "Gemmiger qucibialis"

Metabolome data pre-processing

Filter the data

Similar to above, metabolome features that are rare should be removed. Here, we are filtering out metabolites which are NOT present in more than 50% of the samples.

R 

metab_filt <- filter_metab_abund(x = metab, cutoff = 100, min_prop = 0.5)

OUTPUT 

Kept 217 / 367 features (59.13%) that passed the threshold in ≥ 50% of samples.

Transform the data

Metabolome data here is transformed using.

R 

metab_log <- transform_metab(metab_filt, pseudocount = 1e-6)

dim(metab_log)

OUTPUT 

[1] 144 217

Initial analysis: Unsupervised integration using PLS

Callout

Before training DIABLO, it is important to explore the datasets separately (e.g., PCA, sPLS-DA) and perform unsupervised integration (e.g., rCCA or sPLS) to understand correlation structure.

Here we use sPLS and request 25 variables from each dataset (arbitrary) that maximise covariance between microbiome and metabolome.

R 

# select the number of features to keep in the model
list.keepX = c(25, 25) 
list.keepY = c(25, 25)

# run sPLS
spls_res <- spls(microb_rclr, metab_log, 
             keepX = list.keepX, keepY = list.keepY,
             scale = T)

The correlation circle plot below is a good starting point to visualise the correlations between the selected features for each component, here showing features with |correlation| above 0.5. Points that cluster together are positively associated with each other, whereas points that are on the opposite end are negatively associated with each other.

There is a cluster of microbiome features that are positively correlated with metabolome features on the first component (purple and green dots on the right side of the plot), with a small selection of metabolome with opposite correlations (green dots on the left side of the plot). There is also a cluster of microbiome features that have a contribution towards Component 2, with only 1 (out of the 25) top metabolome features that have some sort of correlative relationship between the two.

R 

# plot features of first PLS
plotVar(spls_res, cutoff = 0.5, title = "(a) microbiome vs metabolomics", 
        legend = c("microbiome", "metabolomics"), 
        var.names = FALSE, style = 'graphics', 
        pch = c(16, 17), cex = c(2,2), 
        col = c('darkorchid', 'lightgreen'))

Below shows the correlation between the top 25 microbiome and metabolome features, with the rows corresponding to microbiome Component 1 and 2, and columns corresponding to metabolomics Component 1 and 2. The first component from the microbiome data shows a very strong correlation (~0.9) with the first component from the metabolomics data, while the other components show much weaker correlations. This suggests that most of the shared signal between the two datasets is captured by the first component.

R 

# calculate correlation of microbiome and metabolomics
cors <- cor(spls_res$variates$X, spls_res$variates$Y)

rownames(cors) <- paste0("microb_comp", 1:nrow(cors))
colnames(cors) <- paste0("metab_comp", 1:ncol(cors))

cors

OUTPUT 

             metab_comp1   metab_comp2
microb_comp1  0.89514137 -2.100475e-17
microb_comp2  0.02162159  7.082259e-01

DIABLO integration

Setup: Split train and test data

To evaluate the ability of DIABLO to generalise the signatures to new datasets, we are using 80% of data (115 samples) to train the DIABLO model to learn discriminative features, and assessing the performance of the model on the remaining 20% of the data (29 samples).

R 

set.seed(123)   # reproducible
index <- sample(1:nrow(microb_rclr), size = 0.8 * nrow(microb_rclr))

# Separate training / testing data
## Microbiome
train_microb <- microb_rclr[index, ]
test_microb  <- microb_rclr[-index, ]

## Metabolomics
train_metab <- metab_log[index, ]
test_metab <- metab_log[-index, ]

## Metadata
train_meta <- meta[index,]
test_meta <- meta[-index,]

# Put data in list
data_diablo = list(microb = train_microb,
                   metab = train_metab)

Setup: Design for DIABLO

DIABLO requires a design matrix (0-1 scale), which represents the strength of the relationship to be modeled between two given dataframes. Values close to 1 prioritise maximising correlations between the datasets; values close to 0 prioritise the discriminative ability of the model. Since predictive ability of the model is desired here, we use a design matrix of 0.1.

R 

## Setup design
design <- matrix(0.1, ncol = length(data_diablo), nrow = length(data_diablo), 
                dimnames = list(names(data_diablo), names(data_diablo)))
diag(design) <- 0
design

OUTPUT 

       microb metab
microb    0.0   0.1
metab     0.1   0.0

With a design in place, the initial DIABLO model can be fitted using an arbitrary number of components (ncomp = 5) before tuning.

R 

# form basic DIABLO model
basic.diablo.model = block.splsda(X = data_diablo, Y = train_meta$Group, ncomp = 5, design = design)

OUTPUT 

Design matrix has changed to include Y; each block will be
            linked to Y.

Tuning parameters

Number of components

To choose the number of components for the final DIABLO model, the function perf() is run with 10-fold cross-validation repeated 10 times, computing performance for each omics block separately.

In the case where you have small number of samples (n < ~10/group), you can use the ‘LOO’ (Leave-one-out) cross-validation method.

R 

# Tune number of components
perf.diablo = perf(basic.diablo.model, validation = 'Mfold', 
                   folds = 10, nrepeat = 10) 

perf.diablo$error.rate

OUTPUT 

$microb
       max.dist centroids.dist mahalanobis.dist
comp1 0.1173913      0.1208696        0.1208696
comp2 0.1191304      0.1217391        0.1191304
comp3 0.1339130      0.1182609        0.1260870
comp4 0.1443478      0.1252174        0.1434783
comp5 0.1434783      0.1252174        0.1434783

$metab
       max.dist centroids.dist mahalanobis.dist
comp1 0.1704348      0.1921739        0.1921739
comp2 0.1530435      0.1521739        0.1539130
comp3 0.1278261      0.1452174        0.1295652
comp4 0.1321739      0.1504348        0.1408696
comp5 0.1286957      0.1469565        0.1295652

The above shows the cross-validated error rates for the microbiome and metabolomics blocks across 1–5 components, evaluated under three different prediction distance metrics (max.dist, centroids.dist, mahalanobis.dist). Lower values indicate better classification performance.

We focus on the centroids.dist as it is a metric that is stable with high-dimensional omics and robust when classes are imbalanced, so it typically gives the most reliable and interpretable performance estimates.

  • For the microbiome block: The error rate reduces slightly moving from Component 1 to Component 2. Increasing the number of components (Components 3–5) give only marginal further reductions.

  • For the metabolome block: Component 1 performs weakest. Increasing to Component 2 improve performance, with the best at Component 3. After Component 3, error does not continue decreasing — Components 4–5 fluctuate slightly, meaning no reliable gain.

The performance plateaus after Component 2–3, and later components provide negligible improvement.

You can visualize the error rates across the datasets as below.

R 

plot(perf.diablo) # plot output of tuning

Centroids distance: First, for each of the classes, the centroid is calculated using all the training samples associated with that class. The Euclidean distance of the test sample to the centroid of training samples are calculated. The class (outcome) will be assigned to that sample based on whichever class centroid is closest to that sample. Classifications made using this metric are less susceptible to outliers within the training set. This metric is best used when the classes cluster moderately well - which can be determined by plotting the samples via the plotIndiv() function.

BER: Average misclassification rate across all classes. It is a metric that gives equal weight to each class, regardless of how many samples are in each class. Perfect prediction will give a BER of 0, whereas a random chance prediction will have BER of 0.5 (for 2 classes (or worse for more classes). Higher values indicates worse performance, whereas lower values indicates better performance.

In DIABLO tuning, adding components is stopped when additional components do not reduce BER in a consistent and meaningful way. Hence, in this case, the tuning results suggest that 1 component is enough to discriminate between the groups. This can occur when a dataset has strong discrminative signals.

WeightedVote.error.rate: Prediction is based on a weighted vote across components, where early components get more weight (because they usually capture more discriminative signal). This tends to work well when signal strength is concentrated in the first component.

Callout

Note: For visualisations (e.g, sample plots), a minimum of 2 components is required. In such cases, set ncomp = 2 in the final DIABLO model, but interpret the biology primarily using features from Component 1.

R 

perf.diablo$choice.ncomp$WeightedVote

OUTPUT 

            max.dist centroids.dist mahalanobis.dist
Overall.ER         1              1                1
Overall.BER        1              1                1

R 

# Set ncomp = 2 for visualisation
ncomp = 2 # else perf.diablo$choice.ncomp$WeightedVote["Overall.BER", "centroids.dist"]

Number of features

DIABLO performs sparse variable selection, retaining only the top number of features in each block with highest covariance while maximising discrimination. To find the optimal number of features to select in each dataset , you can use tune.block.splsda().

Below, we set a range of values to test for the function to test iteratively to find the right number of features with the least error rate (from 5 to 10, and then incrementally by 2 until 20, and then incrementally by 5 until 30). You can set different ranges for the different datasets You can also identify the optimal number of features to select based on previous exploration of each omics data using sPLS-DA.

In this step, DIABLO evaluates each candidate number of selected features using internal cross-validation. For every fold, the model predicts class membership using the centroid-distance rule (dist = "centroids.dist"), and the feature subset that gives the lowest cross-validated error is retained as the optimal keepX.

Here, this is tuned with 10-fold cross validation, but repeated only once because of time constraints. In practice, a more thorough tuning process (with increased nrepeat argument) is recommended. The features are selected based on the centroids dists, on the internal Mfold validation.

R 

# *Can take awhile to run
# set grid of values for each component to test
test.keepX = list (microb = c(5:9, seq(10, 18, 2), seq(20,30,5)), 
                   metab = c(5:9, seq(10, 18, 2), seq(20,30,5)))

tune.diablo = tune.block.splsda(X = data_diablo, 
                                Y = train_meta$Group, ncomp = 2, 
                              test.keepX = test.keepX, design = design,
                              validation = 'Mfold', folds = 10, nrepeat = 1,
                              dist = "centroids.dist")

OUTPUT 

Design matrix has changed to include Y; each block will be
            linked to Y.

OUTPUT 


You have provided a sequence of keepX of length: 13 for block microb and 13 for block metab.
This results in 169 models being fitted for each component and each nrepeat, this may take some time to run, be patient!

OUTPUT 


You can look into the 'BPPARAM' argument to speed up computation time.

The final number of optimal features selected are as below, first and second number for each block corresponding to Component 1 and 2 respectively:

R 

list.keepX = tune.diablo$choice.keepX # set the optimal values of features to retain
list.keepX

OUTPUT 

$microb
[1] 18  5

$metab
[1]  6 10

Final DIABLO model

We then run the final DIABLO model using the parameters from above. A warning message is expected: DIABLO automatically includes the outcome in the design so that each block’s component is associated with the group outcome.

R 

final.diablo.model = block.splsda(X = data_diablo, 
                                  Y = train_meta$Group, 
                                  ncomp = ncomp, 
                                  keepX = list.keepX, design = design)

OUTPUT 

Design matrix has changed to include Y; each block will be
            linked to Y.

Visualisation

Sample plots

The plot below from plotDiablo() is a diagnostic plot that shows how strongly the components extracted from each block are correlated, as specified by the design matrix. The ncomp argument selects which component is shown.

As you can see from the figures below, the first components of the microbiome and metabolome block are highly correlated to each other (indicated by the large correlation value shown in the bottom left). The colours and ellipses related to the sample subtypes indicate the discriminative power of each component to separate controls from CD.

  • On Component 1: the group centroids are clearly separated although the confidence ellipses overlap moderately.
  • On Component 2: the confidence ellipses overlap substantially, suggesting little additional discriminative signal beyond the first component

R 

plotDiablo(final.diablo.model, ncomp = 1)
Component 1
Component 1

R 

plotDiablo(final.diablo.model, ncomp = 2)
Component 2
Component 2

The sample plot below from plotIndiv() function projects each sample into the latent space of each block, allowing visual assessment of clustering patterns. Clustering of the samples can be better assessed with this plot. Here, we can see that both datasets show comparable separation of controls vs CD, suggesting that discriminatory information is present in both omics.

R 

plotIndiv(final.diablo.model, ind.names = FALSE, legend = TRUE, 
          title = 'DIABLO Sample Plots')

The plot below is an arrow plot from plotArrow() function, showing per-sample agreement across datasets. Short, aligned arrows imply high agreement across omics; longer or diverging arrows indicate disagreement.

R 

plotArrow(final.diablo.model, ind.names = FALSE, legend = TRUE, 
          title = 'DIABLO')

Variable plots

The correlation circle plot below shows the relationship between the selected features and their respective components.

On Component 1, a group of microbiome features is positively associated with a set of metabolite features, while another smaller subset of metabolites shows opposite correlations.

On Component 2, a small number of metabolome features correlate with microbiome, however, as shown above, those features are not important in discriminating between the groups so they can be ignored in the interpretation.

R 

plotVar(final.diablo.model, var.names = FALSE, 
        style = 'graphics', legend = TRUE,
        pch = c(16, 17), cex = c(1.5,1.5), 
        col = c('darkorchid', 'lightgreen'))

You can visualize the loading weights of each selected variable on each component and each data set using plotLoadings(). Colours represent the group in which the feature shows the highest expression (contrib = 'max') using the median (method = 'median').

R 

plotLoadings(final.diablo.model, comp = 1, 
             contrib = 'max', method = 'median')

The circos plot below visualises pairwise correlations between selected variables from different omics, represented on the side quadrants. Here, we are visualizing correlations with at least a strength of 0.7 (cutoff parameter), depicted by the lines in the center. The outer most ring indicates the block membership and group-specific expression levels (Control and CD).

R 

circosPlot(final.diablo.model, cutoff = 0.7, line = TRUE,
           color.blocks= c('darkorchid', 'lightgreen'),
           color.cor = c("chocolate3","grey20"), size.labels = 1.5, size.variables = 0.6)

The clustered image map (CIM) below is a heatmap of the values of selected multi-omics signature across samples. By default, Euclidean distance and Complete linkage methods are used.

R 

# *Can run into "figure margins too large" error
cimDiablo(final.diablo.model,margins = c(8, 8))

OUTPUT 


trimming values to [-3, 3] range for cim visualisation. See 'trim' arg in ?cimDiablo

Evaluate model performance

To assess the performance of the model, we use the perf() function to perform 10-fold cross-validation repeated 10 times. In each cross-validation round, for each block in the DIABLO model, a predicted class is obtained for every test sample. These block-specific predictions are then combined to produce a single final prediction per sample, which is used to compute the different error-rate metrics.

MajorityVote: Each block contributes one class vote. The predicted class for a sample is simply the class receiving the highest number of votes across blocks.

Example: with three omics blocks and classes A and B, if the block-level predictions for a sample are A, A, B, the MajorityVote result for that sample is A.

WeightedVote: Each block casts a class vote, but votes are multiplied by block-specific weights that reflect the correlation between the block’s latent component and the outcome. The class whose votes sum to the highest weighted total becomes the predicted class.

Example: Using the previous block-level predictions (A, A, B) and block weights 0.3, 0.2, and 0.7, the weighted votes for class A total 0.3 + 0.2 = 0.5, and the weighted vote for class B is 0.7. Therefore, the WeightedVote result for that sample is B.

R 

# *Can take awhile to run
# run repeated CV performance evaluation
perf.diablo = perf(final.diablo.model, validation = 'Mfold', 
                   M = 10, nrepeat = 10, 
                   dist = 'centroids.dist') 

perf.diablo$MajorityVote.error.rate

OUTPUT 

$centroids.dist
                comp1     comp2
CD          0.2305556 0.2180556
Control     0.1581395 0.1697674
Overall.ER  0.2034783 0.2000000
Overall.BER 0.1943475 0.1939115

R 

perf.diablo$WeightedPredict.error.rate

OUTPUT 

                comp1     comp2
CD          0.1194444 0.1541667
Control     0.1325581 0.1534884
Overall.ER  0.1243478 0.1539130
Overall.BER 0.1260013 0.1538275

From the results above, it can be seen that the error rate is quite low across the board, suggesting good classification performance. Let’s try the model on the test set to see how good it is at classifying novel samples.

Prediction on test set

We now test the model on the held-out samples using the predict() function.

R 

data_test = list(microb = test_microb, 
                 metab = test_metab)

predict_diablo = predict(final.diablo.model, newdata = data_test)

To inspect the model’s prediction accuracy, we are using a confusion matrix.

A confusion matrix is a table that compares a model’s predicted labels with the true labels in a classification problem. It tells you not just how many predictions were wrong, but which classes were confused with each other. This is especially important when:

  • Accuracy alone hides which groups perform poorly
  • Some classes are harder to classify than others
  • There is class imbalance

High diagonal values indicate good agreement between predicted and true labels.

R 

confusion.mat = get.confusion_matrix(truth = test_meta$Group,
                                     predicted = predict_diablo$WeightedVote$centroids.dist[,2])
confusion.mat

OUTPUT 

        predicted.as.CD predicted.as.Control
CD                   14                    2
Control               2                   11

The balanced error rate (BER) remains low, confirming that the DIABLO signature generalises well to the unseen test samples and performs similarly across both classes.

R 

get.BER(confusion.mat)

OUTPUT 

[1] 0.1394231
Key Points
  • DIABLO integrates multiple omics datasets by identifying correlated and discriminative features across blocks.
  • Proper filtering and transformation of each omics dataset are essential for stable multi-omics models.
  • Tuning (components and keepX) guides the selection of the most predictive and biologically relevant features.
  • DIABLO visualisations (sample plots, arrow plots, circos, CIM) reveal relationships across omics and help interpret multi-omics signatures.