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.
Highlights
Until the late 1980s, microbial ecologists and taxonomists relied on culturing and morphological and physiological characteristics to describe microbial communities and members thereof
These results indicate that, on average, a higher operational taxonomic units (OTUs) richness and a better coverage of the fungal community can be expected for the ITS86F/ITS4 and ITS3/ITS4 primer pairs
As most rarefaction curves tended towards saturation, the sequencing depth was assumed to be sufficient to retrieve the most abundant fungal OTUs in analysed soil samples that are detectable by the respective primers and 454 amplicon pyrosequencing
Summary
Until the late 1980s, microbial ecologists and taxonomists relied on culturing and morphological and physiological characteristics to describe microbial communities and members thereof. A number of nextgeneration sequencing technologies enable researchers to identify a large number of organisms from environmental samples using relatively short DNA sequences. This molecular identification method has been termed metabarcoding [6]. Whatever sequencing technology is used, DNA metabarcoding generally depends on the amplification of barcode regions using taxon-specific primers [7] Such primers need to be universal enough to cover a large group of taxa (e.g. the fungal kingdom), but at the same time have to result in amplicons that are variable enough to efficiently distinguish between closely related species or to identify operational taxonomic units (OTUs) [7,8]. The internal transcribed spacer region (ITS; spanning the ITS1, 5.8S and ITS2 regions) in the ribosomal RNA (rRNA) operon has been recognized as the formal DNA barcoding region [9,10,11]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
