Development of Fungal Mock Community Standards for Mycobiome Studies

Most of the initial studies on the human microbiome have been primarily focused on bacterial communities. Given that fungi are ubiquitous and live in symbiosis with the human body, researchers are now actively looking into the role of the mycobiome in human health and disease. Recent advancements in sequencing technologies have enabled the community profiling of fungi; however, the complexities associated with metagenomics sequencing analyses pose significant challenges toward assay standardization. To address this, we have developed two fungal mock community standards consisting of 10 diverse and clinically relevant fungi from the genera Saccharomyces, Malassezia, Candida, Cryptococcus, Cutaneotrichosporon, Trichophyton, Penicillium, Aspergillus, and Fusarium. To enable assay optimization at each step of the workflow—from DNA extraction to data analysis—we developed whole cell– and genomic DNA–based mock microbial communities comprising the same 10 organisms mixed in equal proportions of 2 x 10⁶ cells or genome copies of each organism, respectively. As a proof-of-concept, we used the whole cell mock community to compare two commercially available DNA extraction kits. Here, the extracted DNA was analyzed via ITS and shotgun metagenomics sequencing on the MiSeq^® (Illumina) and MinION^® (Oxford Nanopore Technologies) platforms, and the genomic DNA mock community standard was run in parallel as a control. From our analysis, we found that both the whole cell and genomic DNA mock communities produced similar profiles. Further, using the One Codex database, among other public databases, we were able to identify all 10 organisms to the species level; however, the reported abundance values varied significantly for each organism. Of note, we discovered that Fusarium keratoplasticum (which is part of the F. solani species complex) and the Penicillium and Saccharomyces strains were misidentified by different analysis platforms, thereby demonstrating the inadequate quality and completeness of the existing fungal databases. Overall, our results demonstrate the utility of these mock fungal communities in the standardization and optimization of mycobiome workflows.