Statistical significance threshold application

statistical-significance-threshold-application

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Summary

Apply adjusted p-value (padj) thresholds to metabolomics summary statistics to identify and isolate significantly altered metabolites for downstream analysis and visualization. This skill filters omu_summary output to retain only compounds meeting a user-defined statistical significance criterion, typically padj ≤ 0.05.

When to use

You have generated omu_summary or anova_function output (a dataframe with padj values for all tested metabolites) and need to subset compounds for class-specific frequency counting, fold-change analysis, or visualization. Apply this skill when you want to focus downstream analyses only on metabolites that pass a predefined significance threshold (e.g., padj ≤ 0.05) rather than analyzing the full set of detected compounds.

When NOT to use

• Your analysis goal requires analysis of all detected metabolites regardless of statistical significance (e.g., exploratory profiling of the entire detected metabolome).
• Input data lacks padj or p-value columns (e.g., raw abundance table or untested count data).
• You are performing unsupervised analysis (e.g., PCA) where filtering by significance may bias ordination.

Inputs

• omu_summary output dataframe (or anova_function output)
• Dataframe columns: metabolite names, Class metadata, padj values, log2FoldChange

Outputs

• Filtered dataframe (subset of rows meeting padj ≤ threshold)
• Optionally, frequency table (count_fold_changes output)
• Optionally, relative abundance table (ra_table output)
• Optionally, pie_chart or bar visualization object

How to apply

Load the omu_summary output dataframe containing metabolite names, Class metadata, padj values, and log2FoldChange columns. Use base R subsetting (e.g., subset() or bracket indexing) to filter rows where padj ≤ 0.05 (or your chosen threshold). Optionally, further subset by compound Class (e.g., Class == 'Organic acids') to isolate metabolites of interest. Pass the filtered dataframe to downstream omu functions such as count_fold_changes (with sig_threshold=0.05), ra_table, or pie_chart to generate frequency tables and visualizations. The rationale is that filtering by adjusted p-value controls for multiple testing and ensures only statistically defensible metabolite subsets are retained for interpretation.

Related tools

• omu_summary (Generates univariate statistical test output (t-test or ANOVA) containing padj values and log2FoldChange for all metabolites; output is the primary input to threshold filtering.) — github.com/connor-reid-tiffany/Omu
• count_fold_changes (Accepts filtered (padj-thresholded) omu_summary output and generates frequency tables counting significantly increased vs. decreased metabolites by Class.) — github.com/connor-reid-tiffany/Omu
• ra_table (Transforms count_fold_changes frequency output to relative abundance (percentage) format for visualization-ready input.) — github.com/connor-reid-tiffany/Omu
• pie_chart (Accepts ra_table output (post-threshold) to visualize frequency distribution of significantly altered metabolite classes.) — github.com/connor-reid-tiffany/Omu
• ggplot2 (Rendering engine for omu visualization objects (pie_chart, plot_bar); figures are ggplot2 objects compatible with ggplot2 themes.)
• R (Language and runtime for loading, subsetting, and filtering dataframes using base R indexing and subset() function.)

Examples

filtered_organic_acids <- subset(omu_summary_result, Class == 'Organic acids' & padj <= 0.05); freq_table <- count_fold_changes(filtered_organic_acids, column='Class', sig_threshold=0.05)

Evaluation signals

• Row count of filtered dataframe is ≤ row count of input omu_summary output; all rows in filtered output meet padj ≤ threshold criterion.
• All padj values in filtered output are ≤ the specified threshold (e.g., 0.05); no padj values > threshold remain.
• Downstream count_fold_changes output correctly reflects only the metabolites in the filtered subset (row counts match expected number of increased/decreased compounds).
• Pie chart or bar visualization proportions sum to 100% and include only Classes represented in the filtered subset.
• Log2FoldChange values in filtered output show the expected direction and magnitude of effect (e.g., positive for upregulated, negative for downregulated metabolites).

Limitations

• Threshold selection (e.g., padj ≤ 0.05) is user-defined and may be overly conservative or permissive depending on study design, sample size, and biological context; no guidance is provided in the article for threshold optimization.
• Filtering by padj removes compounds that may be biologically relevant but fail to meet statistical significance, potentially obscuring subtle but consistent metabolic changes.
• Class metadata must be present and correctly assigned (via assign_hierarchy) prior to filtering; missing or incorrect Class values will cause unexpected behavior in downstream functions.
• The article does not discuss multiple testing correction method or provide options for alternative p-value correction schemes beyond the default omu_summary output.

Evidence

• [other] Subset the dataframe to rows where Class == 'Organic acids' and padj <= 0.05 using base R subset and indexing.: "Subset the dataframe to rows where Class == 'Organic acids' and padj <= 0.05 using base R subset and indexing."
• [other] Apply count_fold_changes with column='Class' and sig_threshold=0.05 to generate a frequency table counting increased vs. decreased metabolites.: "Apply count_fold_changes with column='Class' and sig_threshold=0.05 to generate a frequency table counting increased vs. decreased metabolites."
• [other] Omu supports two univariate statistical models, t test and anova, using the functions omu_summary and anova_function respectively: "Omu supports two univariate statistical models, t test and anova, using the functions omu_summary and anova_function respectively"
• [other] The figure is a ggplot2 object, so it is compatible with any ggplot2 themes: "The figure is a ggplot2 object, so it is compatible with any ggplot2 themes"
• [other] Load the omu_summary output dataframe (containing metabolite names, Class metadata, padj values, and log2FoldChange).: "Load the omu_summary output dataframe (containing metabolite names, Class metadata, padj values, and log2FoldChange)."