Utilizing ITS1 and ITS2 to study environmental fungal diversity using pyrosequencing.

The shorter reads generated by high-throughput sequencing has led to a focus on either the ITS1 or the ITS2 sublocus in fungal diversity analyses. Our study aimed to determine how making this choice would influence the datasets obtained and our vision of environmental fungal diversity. DNA was extracted from different environmental samples (water, sediments and soil) and the total internal transcribed spacer (ITS) locus was amplified. 454-sequencing was performed targeting both ITS1 and ITS2. No significant differences in the number of sequences, operational taxonomic units (OTUs) and in the dominant OTUs were detected but less diversity was observed in the ITS2 dataset. In the soil samples, differences in the fungal taxonomic identification were observed, with more Basidiomycota in the ITS1 dataset and more Ascomycota in the ITS2 dataset. Only one-third of the OTUs were detected in both datasets which could be due to (1) more short sequences removed in the ITS2 dataset, (2) different taxonomic affiliation depending on the sublocus used as BLASTn query and/or (3) selectivity in how a primer amplifies the true community. Although ITS1 and ITS2 datasets led to similar results at the fungal community level, for further in-depth diversity analysis this study suggests the analysis of both ITS regions, as they provided different information and were complementary.

Monard C ，Gantner S ，Stenlid J 《-》

New Primers for Discovering Fungal Diversity Using Nuclear Large Ribosomal DNA.

Asemaninejad A ，Weerasuriya N ，Gloor GB ，Lindo Z ，Thorn RG ... -  《PLoS One》

Evaluation of the ribosomal DNA internal transcribed spacer (ITS), specifically ITS1 and ITS2, for the analysis of fungal diversity by deep sequencing.

Yang RH ，Su JH ，Shang JJ ，Wu YY ，Li Y ，Bao DP ，Yao YJ ... -  《PLoS One》

ITS-1 versus ITS-2 pyrosequencing: a comparison of fungal populations in truffle grounds.

Mello A ，Napoli C ，Murat C ，Morin E ，Marceddu G ，Bonfante P ... -  《MYCOLOGIA》

ITS1 versus ITS2 as DNA metabarcodes for fungi.

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.

Blaalid R ，Kumar S ，Nilsson RH ，Abarenkov K ，Kirk PM ，Kauserud H ... -  《-》