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Page 6 of 16 Renzi et al. Microbiome Res Rep 2024;3:2 https://dx.doi.org/10.20517/mrr.2023.27

intergenic spacer (IGS), RNA polymerase II (RPB1 and RPB2), β-tubulin II (TUB2), and the
minichromosome maintenance complex component 7 (MCM7) protein [78-82] . The selection of one or more
reference genes is crucial for standardization and promotion of large-scale investigations, but in some cases,
primer bias in targeted sequencing can be overcome by opting for the shotgun metagenomic approach.

Metagenomic whole genome sequencing
Shotgun metagenomic sequencing allows for a higher taxonomic resolution as it sequences most of the
genomes of every organism present within a sample . This capability not only to identifies the organism
[83]
but also characterizes extended profiles, including antimicrobial resistance, genetic subtypes, metabolism,
and virulence . Despite being a highly effective method for describing pathways and discovering novel
[84]
functions, shotgun metagenomics is significantly more expensive and computationally more intensive than
[85]
amplicon sequencing, depending on sequencing depth .
Moreover, due to its non-specificity, WGS is the most unbiased technique but also the most sensitive to host
DNA contamination, especially in soft tissues and biological fluid samples where host DNA can dominate
the sequenced reads . This sensitivity is a significant concern for the study of mycobiota since fungi
[86]
represent only a small fraction of the total microbial biomass. Achieving adequate sequencing depth is
required to perform the analysis. Currently, it appears that low fungal abundance in human samples is
impeding the broad use of metagenomic WGS in human samples, a finding that is unrelated to DNA
extraction techniques and reflects really low total in vivo fungal abundance .
[87]

The development of high-throughput sequencing techniques has greatly benefited our understanding of
microbial ecology. Nevertheless, the most common methods currently in use, which produce short reads,
often suffer from limited species-level resolution and identification uncertainty. Fortunately, recent
developments in long-read sequencing technologies by PacBio and Oxford Nanopore are enabling the
reconstruction of more complete fungal genomes. These long reads, often exceeding 10 kb in length, can
cover critical genomic regions, including highly repetitive ones [84,88-91] .

Using long-read sequencers, researchers have successfully generated whole genomes of major pathogenic
fungi, often in combination with short-read sequencing, a technique known as hybrid assemblies [92-99] .

Bioinformatics
In metagenomics and metabarcoding analyses, data interpretation is a significant challenge. While these
approaches enhance the objectivity of fungal phylogeny and subsequent accurate identification, they
simultaneously generate ever-growing amounts of sequencing data. Addressing the prompt delivery of the
enormous amount of sequence data available to end user introduces a new challenge.

Databases: need for unification
Thanks to advancements in computational technology and bioinformatics tools, large volumes of data can
now be easily stored, annotated, and accessed remotely with relative ease. As a result, a surplus of nucleotide
sequence databases for fungal studies was created . The strategic value of a database is based on its
[23]
accessibility, through which end users may deposit, save, annotate, and retrieve data. It must be considered
that every database has an intrinsic proclivity to become outdated over time. To maintain useful and
relevant databases for diagnostics and research, a dedicated group of trained professionals is required to
carry out an ongoing and systematic curation. Over the last decade, many online fungal databases have been
established for the mycology research community. However, not all of them have a dedicated team of
curators or an updated maintenance system. Some of the most widely used repositories [Table 2], such as