ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases

Abstract

BackgroundDuring the last 15 years the internal transcribed spacer (ITS) of nuclear DNA has been used as a target for analyzing fungal diversity in environmental samples, and has recently been selected as the standard marker for fungal DNA barcoding. In this study we explored the potential amplification biases that various commonly utilized ITS primers might introduce during amplification of different parts of the ITS region in samples containing mixed templates ('environmental barcoding'). We performed in silico PCR analyses with commonly used primer combinations using various ITS datasets obtained from public databases as templates.ResultsSome of the ITS primers, such as ITS1-F, were hampered with a high proportion of mismatches relative to the target sequences, and most of them appeared to introduce taxonomic biases during PCR. Some primers, e.g. ITS1-F, ITS1 and ITS5, were biased towards amplification of basidiomycetes, whereas others, e.g. ITS2, ITS3 and ITS4, were biased towards ascomycetes. The assumed basidiomycete-specific primer ITS4-B only amplified a minor proportion of basidiomycete ITS sequences, even under relaxed PCR conditions. Due to systematic length differences in the ITS2 region as well as the entire ITS, we found that ascomycetes will more easily amplify than basidiomycetes using these regions as targets. This bias can be avoided by using primers amplifying ITS1 only, but this would imply preferential amplification of 'non-dikarya' fungi.ConclusionsWe conclude that ITS primers have to be selected carefully, especially when used for high-throughput sequencing of environmental samples. We suggest that different primer combinations or different parts of the ITS region should be analyzed in parallel, or that alternative ITS primers should be searched for.

Phylogenetic congruence between subtropical trees and their associated fungi

“…Molecular identification through DNA barcoding of fungi has become an integral and essential part of fungal ecology research and has provided new insights into the diversity and ecology of many different groups of fungi (Anderson & Cairney, 2004; Bellemain et al., 2010). Molecular identification has made it possible to study the ecology of fungi in their dominant, but inconspicuous mycelium stage, and not only by observation of fruiting bodies or selective culturing techniques.…”

“…Molecular identification has made it possible to study the ecology of fungi in their dominant, but inconspicuous mycelium stage, and not only by observation of fruiting bodies or selective culturing techniques. Using high‐throughput sequencing, thousands of sequences can be analyzed from a single environmental sample, enabling researchers to undertake an in‐depth analysis of fungal diversity (Bellemain et al., 2010). However, we realize that although ITS combines the highest resolving power for discriminating closely related species and has a high sequencing success rate across a broad range of fungi (Schoch et al., 2012), it has some methodological limitations as functionally distinct fungi (e.g., pathogenic vs. mutualistic species) may have nearly identical ITS sequences.…”

Rare biosphere exploration using high-throughput sequencing: research progress and perspectives

“…Clarke et al 2014;Liu et al 2013;Ovaskainen et al 2013;van Velzen et al 2012;Toju et al 2012). This type of error is especially acute for those studies focusing on rare taxa (Bellemain et al 2010;Engelbrektson et al 2010). For example, estimates of species richness were highly influenced by primers used: short PCR amplicons (\400 bp) produce higher number of operational taxonomic units (OTUs) than do long ones (Huber et al 2009;Engelbrektson et al 2010).…”

Technical note: use of internal transcribed spacer for ruminal yeast identification in dairy cows

“…For these reasons, identification of yeast species by phenotypic characteristics has been largely replaced by DNA-based methods (Kurtzman, 2006) because they are quick, sensitive and specific (Hayashi et al, 2013). The ITS is a PCR approach validated to be useful for analysis of fungal diversity (Bellemain et al, 2010). ITS is a noncoding ribosomal DNA (rDNA) spacer region located between the 18S and 28S ribosomal RNA (rRNA) genes (Sirohi et al, 2013), and comprises ITS1/ITS2 intergenic sequences with highly conserved 5.8 rRNA in between (Zhang et al, 2015).…”