Issue with unclassified taxa Inflating DA analysis at specified taxonomic levels in ANCOM-BC2

[aimirza]

It seems that when performing differential abundance (DA) analysis at the species level, for example, using ANCOM-BC2, ASVs/OTUs not classified at this level are aggregated into higher taxonomic ranks (e.g., genus or family) and included in the analysis. This significantly increases the number of taxa tested, leading to inflated adjusted p-values due to the multiple comparisons, ultimately reducing sensitivity to detect true differences. Additionally, since ANCOM-BC2 uses each taxon as a potential denominator for comparisons, the inclusion of higher-level taxa distorts the analysis at the species level. This problem is not limited to species; it also affects genus-level analysis and any specified taxonomic level where ASVs/OTUs lack proper classification.

Perhaps filtering out ASVs/OTUs without the target taxonomic classification before running ANCOM-BC2 would address this issue? Importantly, we have observed that removing these unclassified taxa also alters the log-fold changes (LFC) of the remaining taxa, suggesting that their inclusion can significantly impact the DA results. A built-in option to automatically exclude taxa lacking classification at the specified level would help ensure more accurate and meaningful DA analysis.