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Page 2 of 16 Renzi et al. Microbiome Res Rep 2024;3:2 https://dx.doi.org/10.20517/mrr.2023.27

any ecosystem on Earth hosts eukaryotic microorganisms, from extremophiles in geothermal vents to
endophytic fungi in plants to parasites or commensals with the gastrointestinal tracts of animals. In host-
associated microbiomes, microbial eukaryotes implement complex interactions with their hosts: in plants,
[2]
they defend the host against herbivorous organisms and enhance nutrients assimilation ; in animals, they
can metabolize plant compounds in the host’s gastrointestinal systems . However, both plants and animals
[3]
[4,5]
can also be afflicted by microbial eukaryotes . In humans, microbial eukaryotes interact with the host
immune system in intricate ways. The low diversity in microbiomes from industrialized countries reflects
the “extinction” reported for bacterial communities, which is a result of globalization . Beyond host
[6-8]
interactions, microbial eukaryotes are essential to the ecology of aquatic and soil ecosystems, where they
serve as primary producers, symbiotic partners, decomposers, and predators [9,10] .

Fungi constitute the group of eukaryotes with the highest diversity and global distribution. Thanks to a wide
range of morphological, physiological, and ecological features, these organisms have evolved to colonize the
most diverse ecosystems . Within the fungal kingdom, yeasts are not strictly identified, as the term refers
[11]
to a unicellular lifestyle that has evolved several times rather than a taxonomic unit . Yeasts and yeast-like
[12]
fungi are the most prevalent eukaryotic components of the microbiota due to their ubiquity, yet their
abundance and influence are frequently underestimated.

Despite their relevance, eukaryotic microorganisms are generally largely neglected in microbiome
investigations . Traditionally, culture-based techniques have been employed to explore and study
[13]
microbial diversity and to obtain a representative set of isolates based on physicochemical variation.
However, due to intrinsic methodological limitations, this approach has been progressively replaced by
culture-independent ones, although it has been rediscovered and subjected to various refinements in recent
years to enable the capture of a broader spectrum of microorganisms [14,15] . Following the advent of Sanger
sequencing, the use of DNA for the identification of microorganisms has become standard practice,
revolutionizing microbial genotyping and taxonomy [16,17] . The most recent rise of second- and third-
generation sequencing approaches has facilitated the advancement of eukaryotic-specific amplicon
sequencing, which is revolutionizing our understanding of the eukaryotic diversity in host-associated and
environmental microbiomes [18-22] . Like all amplicon-based techniques, this approach can suffer from poor
taxonomic precision and difficulty discriminating between closely related species [23,24] . In contrast, whole
metagenome sequencing captures DNA from the entire pool of species present in a microbiome, including
eukaryotes, without the need for experimental selection. Whole metagenome sequencing data are becoming
predominant in microbiome research because they can be used to assemble unknown genomes, classify
strains, and assess the presence or absence of genes and pathways . These methods are useful for
[25]
identifying bacteria and archaea, but microbiome-associated eukaryotes, such as yeasts, are still difficult to
detect, especially in large metagenome sequencing datasets. One of the main reasons for this issue is that
despite being part of one of the largest branches of the “Tree of Life”, the number of high-quality fungal
target sequences or genomes in curated databases is still significantly lower than that of available bacterial
ones, severely limiting the possibility of properly investigating these organisms.

The aim of this review is to outline the current state of research regarding the techniques and experimental
pipelines for the study of yeast metagenomics, focusing on the currently unresolved methodological
challenges as well as the pros and cons of each different approach.

MYCOBIOME: FOCUS ON YEASTS
The term “mycobiome”, coined in 2009 for a study of fungal communities on salt marsh plants using
[26]
molecular fingerprinting, was then used in 2010 to refer to the human oral mycobiome , Now, it is used to
[27]