Abstract
Identifying fungal profiles from metagenomes is challenging due to an incomplete fungal database and limitations in the understanding of software. Here, we describe a protocol for analyzing the fecal metagenome of cancer patients prior to immunotherapy. We describe steps for raw metagenomic sequencing data retrieval, human genome and contaminants elimination, and assigning taxonomy labels to fungal reads. We then detail measures for assessing fungal alpha diversity and beta diversity, along with differential analysis. For complete details on the use and execution of this protocol, please refer to Xiaowen Huang et al.(1).