Differential abundance analysis of genes and pathways in metagenomics

Hello everyone! I am currently working with WGS samples obtained from human skin for my research project, which involves performing a functional analysis and identifying differentially abundant pathways between healthy and non-healthy samples. I utilized humann 3.0 to identify gene families and pathway abundances, but I am facing challenges in determining the appropriate approach for the analysis.

Can you suggest the most suitable tests to identify the differentially expressed pathways? Should I use abundance as RPK or normalize to relative abundance? What are the optimal methods for visualizing my data?

There are gene and pathway models in anpan. For the gene model using the raw abundance values from humann will be fine. For the pathway model you’ll need to transform your data to log10 abundance (my instinct is that log10 RPK will be preferable to log10 relative abundance, but it should be visually obvious on the plot if the transformation is inappropriate for the model) and also provide overall species abundance (from metaphlan or the like). Both models have visualization tools outlined in the tutorial.