I encountered a problem while performing analysis using the PANTHER website. I run the statistical overrepresentation test for a genelist of 99 genes (GO-Slim BP, Binomial, Bonferroni) in February 2020. My result was Bonferroni count = 1 and a list of several enriched GO terms. Next we run the same analysis with the same settings in July 2020 (two days ago) and the result is dramatically different, the Bonferroni count is 1770 and no enriched terms are found.
I am confused - is the result Bonferroni count = 1 possible? Why these results are so different?
This is how the correction is described on PANTHER website:
The expression data analysis statistics now include a Bonferroni correction for multiple testing. The Bonferroni correction is important because we are performing many statistical tests (one for each pathway, or each ontology term) at the same time. This correction multiplies the single-test P-value by the number of independent tests to obtain an expected error rate.
For pathways, we now correct the reported P-values by multiplying by the number of associated pathways with two or more genes. Some proteins participate in multiple pathways, so the tests are not completely independent of each other and the Bonferroni correction is conservative. For ontology terms, the simple Bonferroni correction becomes extremely conservative because parent (more general) and child (more specific) terms are not independent at all: any gene or protein associated with a child term is also associated with the parent (and grandparent, etc.) terms as well.
To estimate the number of independent tests for an ontology, we count the number of classes with at least two genes in the reference list that are annotated directly to that class (i.e. not indirectly via an annotation to a more specific subclass).