Fungal community analysis by high-throughput sequencing of amplified markers – a user's guide

By Björn D. Lindahl, R. H. Nilsson, Leho Tedersoo, Kessy Abarenkov, Tor Carlsen, R. Kjøller, Urmas Kõljalg, T. Pennanen, S. Rosendahl, J. Stenlid and 1 more. New Phytologist, 2013

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Novel high-throughput sequencing methods outperform earlier approaches in terms of resolution and magnitude. They enable identification and relative quantification of community members and offer new insights into fungal community ecology. These methods are currently taking over as the primary tool to assess fungal communities of plant-associated endophytes, pathogens, and mycorrhizal symbionts, as well as free-living saprotrophs. Taking advantage of the collective experience of six research groups, we here review the different stages involved in fungal community analysis, from field sampling via laboratory procedures to bioinformatics and data interpretation. We discuss potential pitfalls, alternatives, and solutions. Highlighted topics are challenges involved in: obtaining representative DNA/RNA samples and replicates that encompass the targeted variation in community composition, selection of marker regions and primers, options for amplification and multiplexing, handling of sequencing errors, and taxonomic identification. Without awareness of methodological biases, limitations of markers, and bioinformatics challenges, large-scale sequencing projects risk yielding artificial results and misleading conclusions.

Subfields: Cell Biology, Pharmacology, Plant Science

View Paper: https://www.semanticscholar.org/paper/b7e4d6a502e1cf13a304ca9b96bc3a7f96820b9b

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Citations in the corpus

The sooty moulds (2014, background): “…) are no longer separated prior to sequencing by cultivation or subcloning of PCR products ( mostly Intergenic Transcribed Spacer region ribosomal RNA or ITS rRNA gene amplicons ) , but the molecules of interest are only separated in the sequencing process itself ( for reviews see Lindahl et al .”
Plant compartment and biogeography affect microbiome composition in cultivated and native Agave species (2015, methodology): “NMDS ordination for the ITS data set was not performed with UniFrac because of known issues with generating accurate phylogenetic trees from the hypervariable ITS2 sequence data (Lindahl et al., 2013).”
Scraping the bottom of the barrel: are rare high throughput sequences artifacts? (2015, methodology): “Conversely, singleton exclusion may minimally affect community composition (Shade et al., 2012) or multivariate analyses (Gobet et al., 2010; Lindahl et al., 2013).”
Role of Fungi in the Biomineralization of Calcite (2016, background): “Consequently, the poor results usually obtained with DNA-based approaches in the field of fungal biomineralization [135] are more likely due to poor primer specificity and under-representation of fungal sequences in databases [136–138], as well as peculiarities of fungal biology [134] in comparison ”
Top 50 most wanted fungi (2016, positive): “In analogy, for sequence types found only in a single study, some sound skepticism is perhaps in place given the sequence quality-related issues involved in studies based on cloning as well as next-generation sequencing (Hyde et al. 2013; Lindahl et al. 2013; Hughes et al. 2015).”
Machine learning for metagenomics: methods and tools (2015, methodology): “…18S, the large subunit (LSU: 23S/25S/28S) and the highly variable spacers ITS1 and ITS2 of the internal transcribed spacer (ITS) region have also been used, in particular for fungi, higher plants, insects and microalgae (Lindahl et al., 2013; Koetschan et al., 2010; Cuadros-Orellana et al., 2013).”
Habitat models of wood-inhabiting fungi along a decay gradient of Norway spruce logs (2015, methodology): “Potentially chimeric sequences were identified and removed by UCHIME as implemented in MOTHUR (Edgar et al., 2011) referencing the UNITE/INSD database (UNITEv6_sh_dynamic; K~oljalg et al., 2013), and sequences that were part of larger segments were grouped (frag.clust).”
Melanization of mycorrhizal fungal necromass structures microbial decomposer communities (2018, methodology): “Following the recommendation of Lindahl et al. (2013), any cell containing less than four sequence reads was zeroed.”
Global diversity and geography of soil fungi (2014, background): “Because richness estimates are calculated based on the frequency of the rarest species, the reliability of singleton data call into question biologically meaningful extrapolations (11).”