Sequence-based methods for detecting and evaluating the human gut mycobiome

Abstract

We surveyed the fungal microbiota in 16 faecal samples from healthy humans with a vegetarian diet. Fungi were identified using molecular cloning, 454 pyrosequencing and a Luminex analyte-specific reagent (ASR) assay, all targeting the ITS region of the rRNA genes. Fungi were detected in each faecal sample and at least 46 distinct fungal operational taxonomic units (OTUs) were detected, from two phyla - Ascomycota and Basidiomycota. Fusarium was the most abundant genus, followed by Malassezia, Penicillium, Aspergillus and Candida. Commonly detected fungi such as Aspergillus and Penicillium, as well as known dietary fungi Agaricus bisporus and Ophiocordyceps sinensis, are presumed to be transient, allochthonous members due to their abundance in the environment or dietary associations. No single method identified the full diversity of fungi in all samples; pyrosequencing detected more distinct OTUs than the other methods, but failed to detect OTUs in some samples that were detected by cloning and/or ASR assays. ASRs were limited by the commercially available assays, but the potential to design new, optimized assays, coupled with speed and cost, makes the ASR method worthy of further study.