As stated in Parts of a GO annotation, there are some required parts of GO annotations- but there are also some optional pieces. Optional does not mean unimportant!
A very rare piece is the NOT qualifier, which is present in fewer than 11k annotations (there are 7,655,937 annotations in the last GO release). Here’s the NOT in action in AmiGO:
NOT is used to make an explicit note that a gene product has been experimentally demonstrated not to be able to carry out a particular activity or it has been shown to have had a loss of function (e.g. loss of an active site or rapid divergence after a duplication event) over the course of evolution. This is particularly important in cases where associating a GO term with a gene product should be avoided (but might otherwise be made, especially by an automated method). For example, if a protein has sequence similarity to an enzyme (whose activity is GO:nnnnnnn), but has been shown experimentally not to have the enzymatic activity, it is annotated as NOT GO:nnnnnnn. NOT is also used when a cited reference explicitly says (e.g. “our favorite protein is not found in the nucleus”). Qualifying an annotation with the qualifier NOT allows annotators to state that a particular gene product is not associated with a particular GO term. It is used when a GO term might otherwise be expected to apply to a gene product, but an experiment, sequence analysis, etc. proves otherwise.
You can think of the NOT as digital wite-out- it works to make sure an annotation is not propagated. It is is not used for negative or inconclusive experimental results.
Although a rare qualifier, this NOT completely changes the meaning of an annotation. If you’re using annotations for enrichment or are a tool developer, make sure you are acknowledging the Qualifier field. We recommend filtering out any annotation with the NOT qualifier in these cases.