DNA extraction and amplicon production strategies deeply inf luence the outcome of gut mycobiome studies

Abstract

Microbial ecology studies are often performed through extraction of metagenomic DNA followed by amplification and sequencing of a marker. It is known that each step may bias the results. These biases have been explored for the study of bacterial communities, but rarely for fungi. Our aim was therefore to evaluate methods for the study of the gut mycobiome. We first evaluated DNA extraction methods in fungal cultures relevant to the gut. Afterwards, to assess how these methods would behave with an actual sample, stool from a donor was spiked with cells from the same cultures. We found that different extraction kits favour some species and bias against others. In terms of amplicon sequencing, we evaluated five primer sets, two for ITS2 and one for ITS1, 18S and 28S rRNA. Results showed that 18S rRNA outperformed the other markers: it was able to amplify all the species in the mock community and to discriminate among them. ITS primers showed both amplification and sequencing biases, the latter related to the variable length of the product. We identified several biases in the characterisation of the gut mycobiome and showed how crucial it is to be aware of these before drawing conclusions from the results of these studies.

Metagenomic Characterization of Indoor Dust Bacterial and Fungal Microbiota in Homes of Asthma and Non-asthma Patients Using Next Generation Sequencing

“…However, we were very disappointed by the low resolution for species identification of non fungal eukaryotes and were not able to analyze them. Thus, if looking only to fungi, the question of using ITS1-2 or 28S rRNA targets could have been asked, while advantages and limits of each target and even primer sets are still debated (Hanson et al, 2016;De Filippis et al, 2017;Frau et al, 2019). Another option is to use shotgun sequencing for taxon identification and abundance assessment for both prokaryotic and eukaryotic sequences.…”

The gut mycobiome: The overlooked constituent of clinical outcomes and treatment complications in patients with cancer and other immunosuppressive conditions

“…Many commonly used fecal genomic DNA extraction protocols are tailored for extracting bacterial genomic DNA and are often imperfect for extracting fungal genomic DNA in regard to bead size for mechanical lysis, enzymatic lysis buffers, and neutralizing or stabilizing agents [55]. Moreover, different extraction kits favor particular fungal species, are biased against others, and are prone to contamination [56]. Thus, one must carefully consider DNA extraction methods based on whether the study is mycobiome specific, or if one needs to combine bacterial and fungal microbiota analyses.…”

“…Most problems, however, lie in the lack of standardized methods for characterization of the mycobiota. When comparing amplicon sequencing, ITS1, ITS2, 18S, and 28S rRNA give slightly different results [56]. The 18S rRNA typically outperforms other markers in its ability to amplify and discriminate different species; however, because fungal rRNA copy numbers vary, there is a strong bias towards fungi with more copies.…”

“…On the other hand, although the internal transcribed spacer (ITS) region represents the formal fungal barcode, it sometimes provides insufficient resolution to distinguish species. ITS primers show both amplification and sequencing biases related to the variable length of the product [56]. The length of the ITS1 and ITS2 markers vary from 50 bp to several kb.…”

Oral mycobiome identification in atopic dermatitis, leukemia, and HIV patients – a systematic review

“…The mycobiome field is underexploited and there is a bias due to the methods used for its characterization. Amplicon sequencing shows a lack of standardized methods by using different primers that can generate very distinguishable results [40]. Usually, studies focus on analyzing mycobiome in a specific subject or when studying a bacteria-fungi consortium.…”