How is KO Enrichment analysis performed in MicrobiomeAnalyst?

In the Shotgun Data Profiling (SDP) module, KO enrichment analysis is performed by first mapping all uploaded genes to the KO knowledge database. Only genes with KO matches will be kept and used for analysis. Duplicate KO terms will be discarded.

If the uploaded data is a list of KOs , KO enrichment is calculated via over-representation analysis (ORA), using the hypergeometric test. ORA tests if a particular group of KOs are represented more than expected by chance within the uploaded list of genes.

If the uploaded data is a KO abundance table , KO enrichment is calculated via the global test algorithm. Global test evaluates whether a set of genes (i.e. KEGG pathways) is significantly associated with a variable of interest. Compared to ORA, which uses only the total number of KO hits in a pathway, global test considers the gene abundance values and is considered to be more sensitive than ORA. It assumes that if a gene set can be used to predict an outcome of interest, the gene expression patterns per outcome must be different. The global test algorithm is implemented in MicrobiomeAnalyst using the globaltest R package. P-values for both methods are corrected for multiple-testing using the Benjamini and Hochberg’s False-Discovery Rate (FDR).