Fungi and ITS best practices!

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• Wear disposable gloves to prevent contamination.

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• Replace or sterilize sampling tools to avoid cross-contamination.

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• Choose appropriate containers based on sample type.

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• Sample during dry weather to prevent water contamination.

Sampling and storing tips

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• Minimize biological activity by storing samples at cold temperatures.

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• Extract DNA immediately or freeze materials in liquid nitrogen and maintain at

-20C better -80°C.

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• Mix pooled subsamples before freezing to ensure homogeneity.

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• Avoid thawing to prevent sample spoilage and changes in communities.

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• Long-term storage at 4°C may alter fungal diversity.

Chill out: Maintaining cool vibes in post-harvest

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• Mixing It Right: Homogenizing the Material

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• Use bead beating, mortar and pestle, micropestles, or a knife mill for small sample numbers.

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• Weigh the required amount for DNA extraction; store the rest for backup or additional analyses

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• Avoid reaching the full capacity of the DNA extraction kit to prevent liquid absorption or inhibitor co-extraction.

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DNA extraction and beyond: A guided journey from homogenization to amplification

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• Follow protocols elaborated for relevant substrates.

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• CTAB and phenol-chloroform protocols are broadly used for obtaining large quantities of long DNA molecules.

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• Manual methods may require further dilution ahead of PCR to minimize the effect of inhibitors.

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• Commercial kits are time-efficient but cost more compared to manual protocols.

Getting the DNA right: Manual vs. commercial kits

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• Impurities inhibiting PCR can be treated during DNA extraction.

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• Pretreatment, purification kits, or precipitation methods can eliminate inhibitors.

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• Dilution of DNA extracts may be sufficient to overcome PCR inhibition.

Troubleshooting PCR: Tackling impurities

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• The internal transcribed spacer (ITS) region is widely used for fungi.

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• Multiple copy numbers, optimal species-level resolution, and design flexibility.

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• Limitations: Inability to target certain fungi (e.g., Microsporidia), lack of variability in some species-rich genera.

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• Considerations for analysis of specific fungi may require additional taxonomic markers.

The power of ITS region in fungi metabarcoding

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• Sequencing read length limits imposed by NGS platforms 🡪 focus on shorter regions, i.e. ITS1 or ITS2 subregions.

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• Taken separately: Lower taxonomic resolution in these subregions.

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• Important considerations for accurate interpretation of major fungal groups.

Navigating ITS1 and ITS2 subregions

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ITS1 or ITS2

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The results obtained are unequivocal towards ITS1 outperformance to ITS2 in terms of richness and taxonomic coverage. The differential abundance analysis did demonstrate that some taxa were exclusively detected only by ITS2, and vice-versa for ITS1.

ITS1

ITS2

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ITS2 may be more variable and recovers more of the molecular diversity. However, we demonstrate that both ITS1 and ITS2 reveal similar patterns in community structure when analyzed in a community ecology context. 

ITS1

ITS2

ITS1 ITS2

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The results show that any of the ITS or LSU sections we tested provided comparable classification accuracy to the genus level and underscore the need for larger and more diverse classification training sets.

ITS1 ITS2

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ITS86F/ITS4, which amplifies the ITS2 region, was shown to be the most suitable primer pair for the characterization of soil fungal communities with metabarcoding.

ITS2

ITS1

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Between ITS1 and ITS2, differences in the best marker were observed according to the studied ecosystem. While ITS2 is best suited to characterize cheese, wine and fermented meat communities, ITS1 performs better for sourdough bread communities.

ITS2

ITS1

ITS2

ITS1

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Tedersoo L., Bahram M, Zinger L, Nilsson RH, Kennedy PG, Yang T, Anslan S and Mikryukov V (2022). Best Practices in Metabarcoding of Fungi: From Experimental Design to Results. Mol. Ecol. 2022, 31, 2769–2795.

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Gardes M and Bruns T (1993). ITS primers with enhanced specificity for basidiomycetes—application to the identification of mycorrhizae and rusts. Mol. Ecol. 2:113–118.

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White T, Burns T, Lee S, and Taylor J (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, p 315–322. In Innis MA, Gelfand DH, Sninsky JJ, and White TJ (ed), PCR protocols: a guide to methods and applications. Academic Press, San Diego, CA.

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References