This protocol describes the steps used to analyze shotgun sequences deposited in in Integrated Microbial Genomes and Microbiomes (IMG/M ) by using functional anotation evidences. KEGG orthology terms and pathways are used as evidence.The abundance of each of the corresponding biomarker genes is estimated by calculating the proportion of amino acid sequences assigned to the KO of interest (estimated gene copies, assembled and unassembled metagenomes, as retrieved from IMG/M) and normalized with respect to the total number of sequences assigned to KOs in each metagenome.