Analyzing the human gut mycobiome – A short guide for beginners

Thielemann, Nadja; Herz, Michaela; Kurzai, Oliver; Martin, Ronny

doi:10.1016/j.csbj.2022.01.008

Cited by 15 publications

(10 citation statements)

References 94 publications

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“…Previous work suggests that the porcine mycobiome has a pattern of α-diversity that is distinct than that of the human gut bacteriome ( Arfken et al., 2020 ), highlighting the need for more animal mycobiome studies. Fungal sequencing has lagged in part due to difficulties in DNA extraction protocols, primer design, database inaccuracies and missing data ( Huffnagle and Noverr, 2013 ; Huseyin et al., 2017 ; Thielemann et al., 2022 ). Thus, technical studies which determine the most accurate sequencing protocols are needed to reduce biases and incorrect research results.…”

Section: Discussionmentioning

confidence: 99%

Porcine fungal mock community analyses: Implications for mycobiome investigations

Arfken¹,

Frey²,

Carrillo³

et al. 2023

Front. Cell. Infect. Microbiol.

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IntroductionThe gut microbiome is an integral partner in host health and plays a role in immune development, altered nutrition, and pathogen prevention. The mycobiome (fungal microbiome) is considered part of the rare biosphere but is still a critical component in health. Next generation sequencing has improved our understanding of fungi in the gut, but methodological challenges remain. Biases are introduced during DNA isolation, primer design and choice, polymerase selection, sequencing platform selection, and data analyses, as fungal reference databases are often incomplete or contain erroneous sequences.MethodsHere, we compared the accuracy of taxonomic identifications and abundances from mycobiome analyses which vary among three commonly selected target gene regions (18S, ITS1, or ITS2) and the reference database (UNITE - ITS1, ITS2 and SILVA - 18S). We analyze multiple communities including individual fungal isolates, a mixed mock community created from five common fungal isolates found in weanling piglet feces, a purchased commercial fungal mock community, and piglet fecal samples. In addition, we calculated gene copy numbers for the 18S, ITS1, and ITS2 regions of each of the five isolates from the piglet fecal mock community to determine whether copy number affects abundance estimates. Finally, we determined the abundance of taxa from several iterations of our in-house fecal community to assess the effects of community composition on taxon abundance.ResultsOverall, no marker-database combination consistently outperformed the others. Internal transcribed space markers were slightly superior to 18S in the identification of species in tested communities, but Lichtheimia corymbifera, a common member of piglet gut communities, was not amplified by ITS1 and ITS2 primers. Thus, ITS based abundance estimates of taxa in piglet mock communities were skewed while 18S marker profiles were more accurate. Kazachstania slooffiae displayed the most stable copy numbers (83-85) while L. corymbifera displayed significant variability (90-144) across gene regions.DiscussionThis study underscores the importance of preliminary studies to assess primer combinations and database choice for the mycobiome sample of interest and raises questions regarding the validity of fungal abundance estimates.

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Section: Discussionmentioning

confidence: 99%

Porcine fungal mock community analyses: Implications for mycobiome investigations

Arfken¹,

Frey²,

Carrillo³

et al. 2023

Front. Cell. Infect. Microbiol.

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“…The DNA extraction from fungal cells is challenging. Fungi, unlike bacteria, have a cell wall that is usually composed of, ß-1,3-glucan, ß-1,6-glucan, mannans, several glycoproteins and can also contain melanin or a rodlet layer ( Thielemann et al, 2022 ). Thus, an essential step for fungal DNA extraction is efficient cell wall lysis by mechanic methods, like repeated beat-beating, followed by enzymatic cell lysis.…”

Section: Methodologies and Challenges In Neonatal Mycobiome Studiesmentioning

confidence: 99%

It’s time to shed some light on the importance of fungi in neonatal intensive care units: what do we know about the neonatal mycobiome?

Wojciechowska,

Salamon,

Wróblewska-Seniuk

2024

Front. Microbiol.

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The 21st century, thanks to the development of molecular methods, including DNA barcoding, using Sanger sequencing, and DNA metabarcoding, based on next-generation sequencing (NGS), is characterized by flourishing research on the human microbiome. Microbial dysbiosis is perceived as a new pathogenetic factor for neonatal diseases. Fungi are crucial, but neglected, components of the neonatal microbiome, which, despite their low abundance, significantly impact morbidity and mortality rates of premature infants hospitalized in Neonatal Intensive Care Units (NICUs). The neonatal mycobiome’s composition and effect on health remain poorly studied research areas. Our knowledge about neonatal mycobiome, composed of limited genera, is mainly based on research on the bacterial microbiome. We presume it is influenced by clinical factors, including prematurity, antibiotic therapy, and type of delivery. Understanding these risk factors may be useful in prevention strategies against dysbiosis and invasive fungal infections. Despite the methodological challenges resulting from the biology of the fungal cell, this topic is an attractive area of research that may contribute to more effective treatment, especially of newborns from risk groups. In this mini review, we discuss the current state of knowledge, research gaps, study difficulties, and future research directions on the neonatal mycobiome, concerning potential future clinical applications.

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“…While microbiome research has largely focused on bacteria, which comprise more than 99% of the genetic material in the microbiome, viral, fungal, and archaeal microbial components are also of interest. 16 The virome, for example, includes viruses that infect host cells and bacteriophages that target bacteria. Alterations in the virome have been associated with human diseases such as SARS-CoV-2, inflammatory bowel disease, diabetes, and colorectal cancer.…”

Section: Current Approaches To Microbiome Data Analysismentioning

confidence: 99%

“… 17 The fungal microbiota, as with viruses, are affected by host factors such as diet and antibiotic use. 16 Fungi have been shown to exhibit complex interactions with the host and with other members of the microbiome. 18 For example, there is evidence that fungi and bacteria together contribute to early-life immune education, and Saccharomyces boulardii has been used as a probiotic to treat bacterial infections.…”

Section: Current Approaches To Microbiome Data Analysismentioning

confidence: 99%

Multi-omic approaches for host-microbiome data integration

Chetty,

Blekhman

2024

Gut Microbes

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The gut microbiome interacts with the host through complex networks that affect physiology and health outcomes. It is becoming clear that these interactions can be measured across many different omics layers, including the genome, transcriptome, epigenome, metabolome, and proteome, among others. Multi-omic studies of the microbiome can provide insight into the mechanisms underlying host-microbe interactions. As more omics layers are considered, increasingly sophisticated statistical methods are required to integrate them. In this review, we provide an overview of approaches currently used to characterize multi-omic interactions between host and microbiome data. While a large number of studies have generated a deeper understanding of host-microbiome interactions, there is still a need for standardization across approaches. Furthermore, microbiome studies would also benefit from the collection and curation of large, publicly available multi-omics datasets.

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Analyzing the human gut mycobiome – A short guide for beginners

Cited by 15 publications

References 94 publications

Porcine fungal mock community analyses: Implications for mycobiome investigations

Porcine fungal mock community analyses: Implications for mycobiome investigations

It’s time to shed some light on the importance of fungi in neonatal intensive care units: what do we know about the neonatal mycobiome?

Multi-omic approaches for host-microbiome data integration

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