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Fungal diversity
Exploration of fungal diversity in situ has also benefited from next-generation DNA sequencing technologies. The use of high-throughput sequencing techniques has been widely adopted by the fungal ecology community since the first publication of their use in the field in 2009. Similar to exploration of bacterial diversity, these techniques have allowed high-resolution studies of fundamental questions in fungal ecology such as phylogeography, fungal diversity in forest soils, stratification of fungal communities in soil horizons, and fungal succession on decomposing plant litter.

The majority of fungal ecology research leveraging next-generation sequencing approaches involves sequencing of PCR amplicons of conserved regions of DNA (i.e. marker genes) to identify and describe the distribution of taxonomic groups in the fungal community in question, though more recent research has focused on sequencing functional gene amplicons (e.g. Baldrian et al. 2012 ). The locus of choice for description of the taxonomic structure of fungal communities has traditionally been the internal transcribed spacer (ITS) region of ribosomal RNA genes due to its utility in identifying fungi to genus or species taxonomic levels, and its high representation in public sequence databases. A second widely used locus (e.g. Amend et al. 2010, Weber et al. 2013 ), the D1-D3 region of 28S ribosomal RNA genes, may not allow the low taxonomic level classification of the ITS, but demonstrates superior performance in sequence alignment and phylogenetics. In addition, the D1-D3 region may be a better candidate for sequencing with Illumina sequencing technologies. Porras-Alfaro et al. showed that the accuracy of classification of either ITS or D1-D3 region sequences was largely based on the sequence composition and quality of databases used for comparison, and poor-quality sequences and sequence misidentification in public databases is a major concern. The construction of sequence databases that have broad representation across fungi, and that are curated by taxonomic experts is a critical next step.

Next-generation sequencing technologies generate large amounts of data, and analysis of fungal marker-gene data is an active area of research. Two primary areas of concern are methods for clustering sequences into operational taxonomic units by sequence similarity, and quality control of sequence data. Currently there is no consensus on preferred methods for clustering, and clustering and sequence processing methods can have a significant impact on results, especially for the variable-length ITS region. In addition, fungal species vary in intra-specific sequence similarity of the ITS region. Recent research has been devoted to development of flexible clustering protocols that allow sequence similarity thresholds to vary by taxonomic groups, which are supported by well-annotated sequences in public sequence databases.