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.

Internal Transcribed Spacer (ITS) as Dna Barcoding to Identify Fungal Species: a Review

“…It is located in the ribosomal RNA operon. The length ranges from 450 to 750 bp (Beeck et al, 2014). This region exists in two parts, ITS1 and ITS2, which are divided by the 5.8S rDNA.…”

“…The key success of amplifying the targeted DNA barcode region is accurate specific primers. According to Beeck et al (2014), the primers must be universal enough to cover a large group of taxa, while at the same time it should produce amplicons that are varied enough to powerfully distinguish the closely related species. Several taxonspecific primers have been designated to allow a selective amplification of fungal sequences (Figure 2 and Table 1).…”

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

“…Because taxonomic resolution strongly depends on the choice of barcodes (Arfi et al 2012;Schoch et al 2012;Kohout et al 2014), the use of different markers and analysis methods renders studies on fungal biodiversity largely incomparable. In addition, alternative primers targeting the same barcode are known to differ in the recovery of taxa both in silico (Bellemain et al 2010) and in vivo (Tedersoo et al 2010;Op de Beeck et al 2014).…”

High-throughput sequencing of African chikanda cake highlights conservation challenges in orchids

“…Several metabarcoding studies have been published that use nrITS1 or nrITS2 for the identification of fungi (Epp et al 2012;Blaalid et al 2013;Schmidt et al 2013;De Beeck et al 2014). So far the only plant studies to use nrITS in metabarcoding have used nrITS2 to determine the taxonomic composition of pollen collected by honey bees (Galimberti et al 2014;Richardson et al 2015) and species composition in herbal medicines (Cheng et al 2014;Ivanova et al 2016).…”