Shotgun metagenomes and multiple primer pair-barcode combinations of amplicons reveal biases in metabarcoding analyses of fungi
By Leho Tedersoo, Sten Anslan, M. Bahram, Sergei Põlme, T. Riit, I. Liiv, Urmas Kõljalg, V. Kisand, Henrik R. Nilsson, F. Hildebrand and 2 more. 2015
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Rapid development of high-throughput (HTS) molecular identification methods has revolutionized our knowledge about taxonomic diversity and ecology of fungi. However, PCR-based methods exhibit multiple technical shortcomings that may bias our understanding of the fungal kingdom. This study was initiated to quantify potential biases in fungal community ecology by comparing the relative performance of amplicon-free shotgun metagenomics and amplicons of nine primer pairs over seven nuclear ribosomal DNA (rDNA) regions often used in metabarcoding analyses. The internal transcribed spacer (ITS) bar codes ITS1 and ITS2 provided greater taxonomic and functional resolution and richness of operational taxonomic units (OTUs) at the 97% similarity threshold compared to barcodes located within the ribosomal small subunit (SSU) and large subunit (LSU) genes. All barcode-primer pair combinations provided consistent results in ranking taxonomic richness and recovering the importance of floristic variables in driving fungal community composition in soils of Papua New Guinea. The choice of forward primer explained up to 2.0% of the variation in OTU-level analysis of the ITS1 and ITS2 barcode data sets. Across the whole data set, barcode-primer pair combination explained 37.6–38.1% of the variation, which surpassed any environmental signal. Overall, the metagenomics data set recovered a similar taxonomic overview, but resulted in much lower fungal rDNA sequencing depth, inability to infer OTUs, and high
Subfields: Cell Biology, Ecology, Plant Science
View Paper: https://www.semanticscholar.org/paper/a0527252edd8a3c8ea317a8dede3bb6ae4cb4e7d
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Citations in the corpus
- Methods for the extraction, storage, amplification and sequencing of DNA from environmental samples (2018, methodology): “For these reasons, shotgun metagenomic data are now frequently used as the standard method to assess biases in PCR amplification (Tedersoo et al. 2015; Tremblay et al. 2015), on the understanding that less biased amplicon-based approaches will mimic the outputs of metagenomic data analysis more clos”
- Top 50 most wanted fungi (2016, unknown): “We typically encounter them through sequences of the internal transcribed spacer (ITS), the formal fungal barcode (Schoch et al. 2012) and the marker of choice in fungal molecular ecology studies (Lindahl et al. 2013; Tedersoo et al. 2015).”
- The legacy effect of cover crops on soil fungal populations in a cereal rotation (2016, methodology): “Rarefied files were dereplicated and singletons discarded as recommended in Tedersoo et al. (2015), and operational taxonomic units (OTUs) assigned using USEARCH/UPARSE (v7 (Edgar, 2013)) at 97% clustering (Tedersoo et al., 2015); clusters containing fewer than 10 sequences were discarded.”