ITS1 versus ITS2 as DNA metabarcodes for fungi

Abstract

The nuclear ribosomal Internal Transcribed Spacer ITS region is widely used as a DNA metabarcoding marker to characterize the diversity and composition of fungal communities. In amplicon pyrosequencing studies of fungal diversity, one of the spacers ITS1 or ITS2 of the ITS region is normally used. In this methodological study we evaluate the usability of ITS1 vs. ITS2 as a DNA metabarcoding marker for fungi. We analyse three data sets: two comprising ITS1 and ITS2 sequences of known taxonomic affiliations and a third comprising ITS1 and ITS2 environmental amplicon pyrosequencing data. Clustering analyses of sequences with known taxonomy using the bioinformatics pipeline ClustEx revealed that a 97% similarity cut-off represent a reasonable threshold for estimating the number of known species in the data sets for both ITS1 and ITS2. However, no single threshold value worked well for all fungi at the same time within the curated UNITE database, and we found that the Operational Taxonomic Unit (OTU) concept is not easily translated into the level of species because many species are distributed over several clusters. Clustering analyses of the 134 692 ITS1 and ITS2 pyrosequences using a 97% similarity cut-off revealed a high similarity between the two data sets when it comes to taxonomic coverage. Although some groups are under- or unrepresented in the two data sets due to, e.g. primer mismatches, our results indicate that ITS1 and ITS2 to a large extent yield similar results when used as DNA metabarcodes for fungi.

Shotgun metagenomes and multiple primer pair-barcode combinations of amplicons reveal biases in metabarcoding analyses of fungi

“…Although all barcode-primer pair combinations revealed that floristic variables account for the strongest effects in fungal community composition, there were statistically significant primer biases that were not reported in previous studies (Arfi et al 2012;Daghino et al 2012;Blaalid et al 2013). At the level of OTUs, the choice of primer pairs for the ITS1 barcode explained 2% of the community composition, which is less than any environmental predictor group.…”

“…In strong disagreement with Waud et al (2014), however, the ITS2 amplicon is unsuitable for studies of orchid mycorrhizal fungi due to multiple mismatches to Tulasnellaceae in primers located in the 5.8S rDNA. With technological advances in read length and quality, we advocate the use of full ITS region due to a better choice of forward primers and the presence of critical differences in either ITS1 or ITS2 in many pairs of closely related species (Blaalid et al 2013;Kõljalg et al 2013). Our in silico and in vivo results indicate that combining amplicons of ITS1Fngs and ITS1ngs forward primers effectively accounts for biases arising from primer mismatches and the occurrence of introns (Oja et al 2015).…”

“…In particular, high-throughput sequencing (HTS) technologies enable documentation and characterization of hundreds to thousands of species simultaneously from biological samples. These methods have been used successfully to uncover diversity and community composition of fungi in various substrates and localities, including little-studied habitats such as mangroves, aquatic ecosystems, and arctic soils (Arfi et al 2012;Blaalid et al 2013;Livermore and Mattes 2013). Similarly to other amplicon-based methods, HTS techniques are sensitive to PCR and primer biases (Lindahl et al 2013).…”

Rare biosphere exploration using high-throughput sequencing: research progress and perspectives

“…Selection of candidate gene regions, as well as associated versatile primers, represents the first most crucial step (e.g. Blaalid et al 2013;Zhan et al 2014a). Versatile primers in the paper refer to PCR primers that can be used to successfully amplify all species members in communities of interest.…”

“…Leray et al 2013;Gibson et al 2014). More successful examples have been reported for the rDNA region, including the internal transcribed spacer (ITS), SSU rDNA, and large subunit ribosomal DNA (LSU rDNA), for diverse communities such as microbes (Nossa et al 2010), fungi (Ihrmark et al 2012;Blaalid et al 2013) and plankton (Zhan et al 2014a).…”