Fungal identification biases in microbiome projects

Tedersoo, Leho; Lindahl, Björn D.

doi:10.1111/1758-2229.12438

Cited by 177 publications

(150 citation statements)

References 39 publications

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“…Alternatively, we opted to compare the taxonomic diversity as far as possible and to comment on the differential detection of taxa. Our approach, which considers the lichen thallus as distinctive and still largely unexplored niche for unknown fungal assemblages, further strengthens the perception that diversity estimates based on metabarcoding are limited by the barcode locus selected (Tedersoo et al 2015; Tedersoo and Lindahl 2016). …”

Section: Discussionsupporting

confidence: 66%

“…They have been carried out on both Ascomycota (Nilsson et al 2009; Bellemain et al 2013) and Basidiomycota (Badotti et al 2017). Taxonomic bias can be introduced by the choice of primers, as these cause a higher number of mismatches in different taxa (Bellemain et al 2013; Tedersoo et al 2015; Tedersoo and Lindahl 2016). Some studies also reported that the two spacers are prone to preferential amplification at different levels (Nilsson et al 2009; Mello et al 2011; Bellemain et al 2013; Bazzicalupo et al 2013; Monard et al 2013).…”

Section: Introductionmentioning

confidence: 99%

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ITS2 metabarcoding analysis complements lichen mycobiome diversity data

et al. 2018

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Lichen thalli harbor complex fungal communities (mycobiomes) of species with divergent trophic and ecological strategies. The complexity and diversity of lichen mycobiomes are still largely unknown, despite surveys combining culture-based methods and high-throughput sequencing (HTS). The results of such surveys are strongly influenced by the barcode locus chosen, its sensitivity in discriminating taxa, and the depth to which public sequence repositories cover the phylogenetic spectrum of fungi. Here, we use HTS of the internal transcribed spacer 2 (ITS2) to assess the taxonomic composition and diversity of a well-characterized, alpine rock lichen community that includes thalli symptomatically infected by lichenicolous fungi as well as asymptomatic thalli. Taxa belonging to the order Chaetothyriales are the major components of the observed lichen mycobiomes. We predict sequences representative of lichenicolous fungi characterized morphologically and assess their asymptomatic presence in lichen thalli. We demonstrated the limitations of metabarcoding in fungi and show how the estimation of species diversity widely differs when ITS1 or ITS2 are used as barcode, and particularly biases the detection of Basidiomycota. The complementary analysis of both ITS1 and ITS2 loci is therefore required to reliably estimate the diversity of lichen mycobiomes.Electronic supplementary materialThe online version of this article (10.1007/s11557-018-1415-4) contains supplementary material, which is available to authorized users.

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Section: Discussionsupporting

confidence: 66%

Section: Introductionmentioning

confidence: 99%

ITS2 metabarcoding analysis complements lichen mycobiome diversity data

et al. 2018

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“…Furthermore, these results indicate that the ITS2 subregion shows either more than two‐fold better resolution for OTU separation at the 97% similarity threshold or accommodates more spurious OTUs. Previously, we attributed the greater richness of ITS2 fungal OTUs to lower primer bias (Tedersoo et al ., ,b; Tedersoo & Lindahl, ), which is minimized here by the use of degenerate ITS2 primer variants (see below). Other studies have observed comparable richness profiles for ITS1 and ITS2 subregions (e.g.…”

Section: Resultsmentioning

confidence: 99%

“…Of the forward primers, the universal 1389F primer performed best by amplifying all fungal phyla and various groups of non‐fungal eukaryotes that contributed 16% of all sequences on average. The ITS9MUNngs primer (5′‐TACACACCGCCCGTCG‐3′; Tedersoo & Lindahl, ) 2 bp downstream has a terminal mismatch to Archaea and Bacteria, but similar affinity to eukaryotes, and can be used alternatively.…”

Section: Resultsmentioning

confidence: 99%

PacBio metabarcoding of Fungi and other eukaryotes: errors, biases and perspectives

2017

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Second-generation, high-throughput sequencing methods have greatly improved our understanding of the ecology of soil microorganisms, yet the short barcodes (< 500 bp) provide limited taxonomic and phylogenetic information for species discrimination and taxonomic assignment. Here, we utilized the third-generation Pacific Biosciences (PacBio) RSII and Sequel instruments to evaluate the suitability of full-length internal transcribed spacer (ITS) barcodes and longer rRNA gene amplicons for metabarcoding Fungi, Oomycetes and other eukaryotes in soil samples. Metabarcoding revealed multiple errors and biases: Taq polymerase substitution errors and mis-incorporating indels in sequencing homopolymers constitute major errors; sequence length biases occur during PCR, library preparation, loading to the sequencing instrument and quality filtering; primer-template mismatches bias the taxonomic profile when using regular and highly degenerate primers. The RSII and Sequel platforms enable the sequencing of amplicons up to 3000 bp, but the sequence quality remains slightly inferior to Illumina sequencing especially in longer amplicons. The full ITS barcode and flanking rRNA small subunit gene greatly improve taxonomic identification at the species and phylum levels, respectively. We conclude that PacBio sequencing provides a viable alternative for metabarcoding of organisms that are of relatively low diversity, require > 500-bp barcode for reliable identification or when phylogenetic approaches are intended.

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“…In conclusion, our results contribute to the evidence that fungal primers for the ITS rDNA, which is the preferred fungal barcode (Lindahl et al ., ), fail to provide proper coverage of AMF diversity, as they do for other important fungal groups (Bellemain et al ., ; Ihrmark et al ., ; Tedersoo and Lindahl, ). Nonetheless, the specific ITS2 primer pair we used in this case study can provide enough data to estimate community structure without major biases and to make reliable conclusions on AMF relation to environmental variables.…”

Section: Discussionmentioning

confidence: 99%

ITS fungal barcoding primers versus 18S AMF‐specific primers reveal similar AMF‐based diversity patterns in roots and soils of three mountain vineyards

Berruti

Desirò

Visentin

et al. 2017

Environ Microbiol Rep

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ITS primers commonly used to describe soil fungi are flawed for AMF although it is unknown the extent to which they distort the interpretation of community patterns. Here, we focus on how the use of a specific ITS2 fungal barcoding primer pair biased for AMF changes the interpretation of AMF community patterns from three mountain vineyards compared to a novel AMF-specific approach on the 18S. We found that although discrepancies were present in the taxonomic composition of the two resulting datasets, the estimation of diversity patterns among AMF communities was similar and resulted in both primer systems being able to correctly assess the community-structuring effect of location, compartment (root vs. soil) and environment. Both methodologies made it possible to detect the same alpha-diversity trend among the locations under study but not between root and soil transects. We show that the ITS2 primer system for fungal barcoding provides a good estimate of both AMF community structure and relation to environmental variables. However, this primer system does not fit in with cross-compartment surveys (roots vs. soil) as it can underestimate AMF diversity in soil samples. When specifically focusing on AMF, the 18S primer system resulted in wide coverage and marginal non-target amplification.

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Fungal identification biases in microbiome projects

Cited by 177 publications

References 39 publications

ITS2 metabarcoding analysis complements lichen mycobiome diversity data

ITS2 metabarcoding analysis complements lichen mycobiome diversity data

PacBio metabarcoding of Fungi and other eukaryotes: errors, biases and perspectives

ITS fungal barcoding primers versus 18S AMF‐specific primers reveal similar AMF‐based diversity patterns in roots and soils of three mountain vineyards

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