OCEAN: a method that defines metabolic enzyme footprint from a curated reduced version of the recon2 reaction network. The metabolic enzyme footprints are used to explore coordinated deregulations of metabolite abundances with respect to their position relative to metabolic enzymes. This is similar to Kinase-substrate and TF-targets enrichment analyses.
Differential analysis
With data from: https://www.embopress.org/doi/full/10.15252/msb.20209730.
unique(toy_targets$condition)
comparisons <- list('tumorVsHealthy' = c(1,-2))
limmaRes <- runLimma(measurements = toy_metabolomic_data,
targets = toy_targets,
comparisons = comparisons)This differential anaylsis result represents metabolic up and down-regulations of metabolite abundances in kidney tumor tissue compared to adjacent non-tumoral tissue.
Format differential analysis result
In order to assess the metabolic imbalance, we use the t statistic of the differential analysis. The t-statistic represents a good estimate of the relative significance of up and down-regulations of metabolic abundances.
t_table <- ttop_list_to_t_table(
limma_res_to_ttop_list(limma_res = limmaRes,
comp_names = names(comparisons),
number = length(toy_metabolomic_data[,1]),
adjust.method = "fdr"))Next, the metabolic names have to be converted to a standard identifer.
In this case, we use KEGG IDs. This step is particularly important because this is where the users metabolic identifiers are mapped to the kegg ids used by the method.
THE USER SHOULD PROVIDE A MAPPING TABLE IN THE SAME FORMAT AS THE ONE PRESENTED HERE. (You can look at it to inspire yourself from it.) The mapping table should map the users own metabolic identifiers to kegg compound IDs.
t_table <- t_table_metactivity_input_formater(
metabolomic_t_table = t_table,
mapping_table = mapping_table,
affixes = c("c","l","x","m","e","n","r")
)Subnetworks
data(expressed_genes)In this case, we will use all pathways available. Users can restrict this list on a per-need basis.
all_pathways <- unique(recon2_redhuman$pathway)
sub_network <- model_to_pathway_sif(pathway_to_keep = all_pathways$X1)Some formating of the network is required, especially to make the method easier to run and the results easier to interpret.
This step simply translate the gene IDs in the enzyme complexes into gene symbols:
sub_network <- translate_complexes(sub_network)Co-factors are removed. since we are aiming to follow metabolic routes in the metabolic reaction network, it is crucial to remove over-promiscuous metabolites that create unnecessary complexity in the network.
sub_network_nocofact <- remove_cofactors(sub_network)Filter out any gene that isn’t expressed from the metabolic network:
non_expressed_genes <- expressed_genes[rowSums(expressed_genes[,c(2),drop = F]) == 0,c(1),drop = F]
non_expressed_genes <- c(non_expressed_genes$gene)
tokeep <- list()
for(i in 1:length(sub_network_nocofact$reaction_network[,1]))
{
elements <- gsub("[><].*","",sub_network_nocofact$reaction_network[i,])
elements <- elements[!grepl("cpd:",elements)]
elements <- unlist(strsplit(elements, "_"))
tokeep[[i]] <- sum(elements %in% non_expressed_genes) == 0
}
tokeep <- unlist(tokeep)
sub_network_nocofact$reaction_network <- sub_network_nocofact$reaction_network[tokeep,]Compress transporters to simplify the network:
sub_network_nocofact <- compress_transporters(sub_network_nocofact = sub_network_nocofact)Split transaminases to preserve correct metabolic transformation routes:
sub_network_nocofact <- split_transaminases(sub_network_nocofact = sub_network_nocofact)
enzymes <- unique(sub_network_nocofact$attributes$V1)
enzymes <- enzymes[!grepl("_[clxmenr]$",enzymes)]Convert the network into a forest (list of enzymes (trees) with correspoding metabolic signatures (branches)):
sub_forest <- forestMaker(enzymes, sub_network_nocofact$reaction_network, branch_length = c(1,1), remove_reverse = T)Prepare the metabolic enzyme sets
penalty_min <- 6 #minimum 1 and integer
penalty_max <- 8 #maximum 9 and integer
reaction_set_list <- prepare_metabolite_set(
penalty_range = penalty_min:penalty_max,
forest = sub_forest,
measured_metabolites = t_table$KEGG
)
reaction_set_list_merged <- condense_metabolite_set(reaction_set_list = reaction_set_list)
penalty <- 8 #has be between penalty_min and penalty_max and integer
regulons_df <- prepare_regulon_df(
reaction_set_list_merged,
penalty,
filter_imbalance = c(0,1)
)Compute metabolic enzme enrichment score
metactivity_res <- metactivity(metabolomic_t_table = t_table,
regulons_df = regulons_df,
compartment_pattern = "_[a-z]$",
k = 1000)
mean_ES_df <- metactivity_res$ES
mean_NES_df <- metactivity_res$NEStranslated_results <- translate_results(regulons_df = regulons_df, t_table = t_table, mapping_table = mapping_table, compress_compartments = T)Visualise results for single enzymes
plots <- plotMetaboliteContribution(
enzyme = 'OGDH_DLD_PDH_DLST>1052',
stat_df = translated_results$t_table,
metabolite_sets = translated_results$regulons_df,
contrast_index = 1,
stat_name = 'Abundance Down <==> Up (t-value)',
scaling_factor = 6,
nLabels = 25
)
plot(plots$scatter)
plots <- plotMetaboliteContribution(
enzyme = 'BCAT1',
stat_df = translated_results$t_table,
metabolite_sets = translated_results$regulons_df,
contrast_index = 1,
stat_name = 'Abundance Down <==> Up (t-value)',
scaling_factor = 6,
nLabels = 25
)
plot(plots$scatter)## R version 4.2.2 (2022-10-31)
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## attached base packages:
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## loaded via a namespace (and not attached):
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