title: Comparison and validation of some ITS primer pairs useful for fungal metabarcoding studies.
abstract: Current metabarcoding studies aiming to characterize microbial communities generally rely on the amplification and sequencing of relatively short DNA regions. For fungi, the internal transcribed spacer (ITS) region in the ribosomal RNA (rRNA) operon has been accepted as the formal fungal barcode. Despite an increasing number of fungal metabarcoding studies, the amplification efficiency of primers is generally not tested prior to their application in metabarcoding studies. Some of the challenges that metabarcoding primers should overcome efficiently are the amplification of target DNA strands in samples rich in non-target DNA and environmental pollutants, such as humic acids, that may have been co-extracted with DNA. In the current study, three selected primer pairs were tested for their suitability as fungal metabarcoding primers. The selected primer pairs include two primer pairs that have been frequently used in fungal metabarcoding studies (ITS1F/ITS2 and ITS3/ITS4) and a primer pair (ITS86F/ITS4) that has been shown to efficiently amplify the ITS2 region of a broad range of fungal taxa in environmental soil samples. The selected primer pairs were evaluated in a 454 amplicon pyrosequencing experiment, real-time PCR (qPCR) experiments and in silico analyses. Results indicate that experimental evaluation of primers provides valuable information that could aid in the selection of suitable primers for fungal metabarcoding studies. Furthermore, we show that the ITS86F/ITS4 primer pair outperforms other primer pairs tested in terms of in silico primer efficiency, PCR efficiency, coverage, number of reads and number of species-level operational taxonomic units (OTUs) obtained. These traits push the ITS86F/ITS4 primer pair forward as highly suitable for studying fungal diversity and community structures using DNA metabarcoding.
authors.affiliation: Centre for Environmental Sciences, Hasselt University, Hasselt, Limburg, Belgium.
authors.forename: Michiel
authors.initials: M
authors.lastname: Op De Beeck
authors.affiliation: Department of Microbial and Molecular Systems (M2S), Catholic University of Leuven, Antwerp, Belgium.
authors.forename: Bart
authors.initials: B
authors.lastname: Lievens
authors.affiliation: Department of Microbial and Molecular Systems (M2S), Catholic University of Leuven, Antwerp, Belgium.
authors.forename: Pieter
authors.initials: P
authors.lastname: Busschaert
authors.affiliation: Earth & Life Institute, Catholic University of Louvain, Louvain-la-Neuve, Walloon Brabant, Belgium.
authors.forename: Stéphan
authors.initials: S
authors.lastname: Declerck
authors.affiliation: Centre for Environmental Sciences, Hasselt University, Hasselt, Limburg, Belgium.
authors.forename: Jaco
authors.initials: J
authors.lastname: Vangronsveld
authors.affiliation: Centre for Environmental Sciences, Hasselt University, Hasselt, Limburg, Belgium.
authors.forename: Jan V
authors.initials: JV
authors.lastname: Colpaert
mesh_terms: D001530:Belgium; D003198:Computer Simulation; D058893:DNA Barcoding, Taxonomic / Q000379:methods*; D017931:DNA Primers / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics*; D005658:Fungi / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012988:Soil Microbiology
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal; D021903:DNA, Ribosomal Spacer