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.

[1]  T. White.  Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics , 1990 .

[2]  T. Bruns,et al.  ITS primers with enhanced specificity for basidiomycetes ‐ application to the identification of mycorrhizae and rusts , 1993, Molecular ecology.

[3]  Erko Stackebrandt,et al.  Taxonomic Note: A Place for DNA-DNA Reassociation and 16S rRNA Sequence Analysis in the Present Species Definition in Bacteriology , 1994 .

[4]  Gapped BLAST and PSI-BLAST: A new , 1997 .

[5]  E. Larsson,et al.  High phylogenetic diversity among corticioid homobasidiomycetes. , 2004, Mycological research.

[6]  A. Jumpponen.  Soil Fungal Communities Underneath Willow Canopies on a Primary Successional Glacier Forefront: rDNA Sequence Results Can Be Affected by Primer Selection and Chimeric Data , 2007, Microbial Ecology.

[7]  James R. Knight,et al.  Genome sequencing in microfabricated high-density picolitre reactors , 2005, Nature.

[8]  Rytas Vilgalys,et al.  Fungal Community Analysis by Large-Scale Sequencing of Environmental Samples , 2005, Applied and Environmental Microbiology.

[9]  Steven L. Miller,et al.  Perspectives in the new Russulales , 2006, Mycologia.

[10]  Adam Godzik,et al.  Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences , 2006, Bioinform..

[11]  J. Horton,et al.  Seasonal dynamics of ectomycorrhizal fungus assemblages on oak seedlings in the southeastern Appalachian Mountains , 2008, Mycorrhiza.

[12]  R. Henrik Nilsson,et al.  Intraspecific ITS Variability in the Kingdom Fungi as Expressed in the International Sequence Databases and Its Implications for Molecular Species Identification , 2008, Evolutionary bioinformatics online.

[13]  J. Guarro,et al.  Sequence-Based Identification of Aspergillus, Fusarium, and Mucorales Species in the Clinical Mycology Laboratory: Where Are We and Where Should We Go from Here? , 2008, Journal of Clinical Microbiology.

[14]  C. Bledsoe,et al.  Contrasting ectomycorrhizal fungal communities on the roots of co-occurring oaks (Quercus spp.) in a California woodland. , 2008, The New phytologist.

[15]  F. Martin,et al.  454 Pyrosequencing analyses of forest soils reveal an unexpectedly high fungal diversity. , 2009, The New phytologist.

[16]  Björn Canbäck,et al.  RAMI: a tool for identification and characterization of phylogenetic clusters in microbial communities , 2009, Bioinform..

[17]  Erik Kristiansson,et al.  An outlook on the fungal internal transcribed spacer sequences in GenBank and the introduction of a web-based tool for the exploration of fungal diversity. , 2009, The New phytologist.

[18]  Erik Kristiansson,et al.  The ITS region as a target for characterization of fungal communities using emerging sequencing technologies. , 2009, FEMS microbiology letters.

[19]  Taylor Mullineux,et al.  Evolution of rDNA ITS1 and ITS2 sequences and RNA secondary structures within members of the fungal genera Grosmannia and Leptographium. , 2009, Fungal genetics and biology : FG & B.

[20]  Surendra Kumar,et al.  AIR: A batch-oriented web program package for construction of supermatrices ready for phylogenomic analyses , 2009, BMC Bioinformatics.

[21]  K. Jones,et al.  Massively parallel 454 sequencing indicates hyperdiverse fungal communities in temperate Quercus macrocarpa phyllosphere. , 2009, The New phytologist.

[22]  C. Quince,et al.  Accurate determination of microbial diversity from 454 pyrosequencing data , 2009, Nature Methods.

[23]  A. Keshavarzian,et al.  Quantitative Assessment of the Human Gut Microbiome Using Multitag Pyrosequencing , 2010, Chemistry & biodiversity.

[24]  R. Henrik Nilsson,et al.  An open source software package for automated extraction of ITS1 and ITS2 from fungal ITS sequences for use in high-throughput community assays and molecular ecology , 2010 .

[25]  R. Henrik Nilsson,et al.  PlutoF—a Web Based Workbench for Ecological and Taxonomic Research, with an Online Implementation for Fungal ITS Sequences , 2010, Evolutionary Bioinformatics Online.

[26]  L. Tedersoo,et al.  454 Pyrosequencing and Sanger sequencing of tropical mycorrhizal fungi provide similar results but reveal substantial methodological biases. , 2010, The New phytologist.

[27]  B. Lindahl,et al.  Production of ectomycorrhizal mycelium peaks during canopy closure in Norway spruce forests. , 2010, The New phytologist.

[28]  Andy F. S. Taylor,et al.  The UNITE database for molecular identification of fungi--recent updates and future perspectives. , 2010, The New phytologist.

[29]  Robert C. Edgar,et al.  BIOINFORMATICS APPLICATIONS NOTE , 2001 .

[30]  Pierre Taberlet,et al.  ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases , 2010, BMC Microbiology.

[31]  Surendra Kumar,et al.  CLOTU: An online pipeline for processing and clustering of 454 amplicon reads into OTUs followed by taxonomic annotation , 2011, BMC Bioinformatics.

[32]  K. I. Ugland,et al.  High diversity of root associated fungi in both alpine and arctic Dryas octopetala , 2010, BMC Plant Biology.

[33]  Priscila Chaverri,et al.  Species delimitation in fungal endophyte diversity studies and its implications in ecological and biogeographic inferences , 2011, Molecular ecology.

[34]  M. Wagner,et al.  Barcoded Primers Used in Multiplex Amplicon Pyrosequencing Bias Amplification , 2011, Applied and Environmental Microbiology.

[35]  H. Kauserud,et al.  High consistency between replicate 454 pyrosequencing analyses of ectomycorrhizal plant root samples , 2012, Mycorrhiza.

[36]  C. Napoli,et al.  ITS-1 versus ITS-2 pyrosequencing: a comparison of fungal populations in truffle grounds , 2011, Mycologia.

[37]  G. Cochrane,et al.  The International Nucleotide Sequence Database Collaboration , 2011, Nucleic Acids Res..

[38]  R. Halvorsen,et al.  Seasonal trends in the biomass and structure of bryophyte-associated fungal communities explored by 454 pyrosequencing. , 2012, The New phytologist.

[39]  Guy Cochrane,et al.  The International Nucleotide Sequence Database Collaboration , 2011, Nucleic Acids Res..

[40]  Daniel L. Lindner,et al.  Don't make a mista(g)ke: is tag switching an overlooked source of error in amplicon pyrosequencing studies? , 2012 .

[41]  Rasko Leinonen,et al.  The sequence read archive: explosive growth of sequencing data , 2011, Nucleic Acids Res..

[42]  John L. Spouge,et al.  Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi , 2012, Proceedings of the National Academy of Sciences.

[43]  L. Zinger,et al.  Two decades of describing the unseen majority of aquatic microbial diversity , 2012, Molecular ecology.

[44]  Anthony Levasseur,et al.  Multiple markers pyrosequencing reveals highly diverse and host-specific fungal communities on the mangrove trees Avicennia marina and Rhizophora stylosa. , 2012, FEMS microbiology ecology.

[45]  K. I. Ugland,et al.  Changes in the root‐associated fungal communities along a primary succession gradient analysed by 454 pyrosequencing , 2012, Molecular ecology.

[46]  P. Taberlet,et al.  Environmental DNA , 2012, Molecular ecology.