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

By E. Bellemain, Tor Carlsen, C. Brochmann, É. Coissac, P. Taberlet, Havard Kauserud. BMC Microbiology, 2010

1,083 citations recorded by the source corpus.

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.

Subfields: Cell Biology, Ecology, Evolution, Behavior and Systematics, Plant Science

View Paper: https://www.semanticscholar.org/paper/e5e11c2e56b8793d4c31fa395d70313e17bdfe58

How researchers see this paper

The researchers whose trusted networks rank it highest, strongest advocate first. Every researcher gets a different order.

In Cell Biology

In Ecology, Evolution, Behavior and Systematics

In Plant Science

Citations in the corpus

Enterotypes of the human gut mycobiome (2022, background): “Such a difference observed for the fourth enterotype between ITS1 and ITS2 can be attributed to different amplicon-targeted regions by ITS1 and ITS2 [35], and we found that Asco-mycota.sp and Saccharomycetales .sp were enriched in ITS1-and ITS2-sequencing datasets ( p < 0.05 with Wilcoxon rank-sum…”
Next‐generation monitoring of aquatic biodiversity using environmental DNA metabarcoding (2016, methodology): “The PCR products were purified and sequenced (forward and reverse) using Sanger technology at the Eurofins MWG Operon sequencing facilities (Ebersberg, Germany).”
Barcoding diatoms: evaluation of the V4 subregion on the 18S rRNA gene, including new primers and protocols (2011, background): “Furthermore, studies in fungi using ITS suggested that errors in amplification/sequencing— especially in high throughput—could easily lead to overestimation of diversity in environmental samples (Bellemain et al. 2010).”
Improved software detection and extraction of ITS1 and ITS2 from ribosomal ITS sequences of fungi and other eukaryotes for analysis of environmental sequencing data (2013, background): “Complications include primer bias (Bellemain et al. 2010), diﬀering evolutionary rates in diﬀerent fungal lineages (Nilsson et al. 2008) and the presence of several diﬀerent copies within a single individual (Lindner et al. 2013).”
Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi (2012, background): “However, all primer sets have a range of biases, and an appropriate solution will be to use more than one primer combination (47).”
Next-Generation Sequencing Technology: Current Trends and Advancements (2023, methodology): “Quality check of sequences FastQC [90], FASTX-toolkit [91], MultiQC [92] Trimming of adaptors and low-quality bases Trimmomatic [93], Cutadapt [94], fastp [95] Alignment of sequence reads to reference genome BWA [96], Bowtie [97], dragMAP [98]”
Long-term and legacy effects of manure application on soil microbial community composition (2018, methodology): “Briefly, for the first PCR reaction, the V4 hypervariable region of the prokaryotic (bacterial and archaeal) 16S rRNA gene was amplified using the primer set 515F ( G TG CCAGCMGCCGCGG TAA ) a n d 8 0 6 R (GGACTACHVGGGTWTCTAAT) (Caporaso et al. 2011), and the internal transcribed spacer region 1 (ITS”
Who is eating what: diet assessment using next generation sequencing (2012, background): “2010) or as internal transcribed spacers (ITS) for fungi (Bellemain et al. 2010).”
High-Coverage ITS Primers for the DNA-Based Identification of Ascomycetes and Basidiomycetes in Environmental Samples (2012, methodology): “Nonetheless, inspection of sequences in the present data sets revealed that concerning the ‘‘non-Dikarya’’ mycorrhizal fungal phylum Glomeromycota, ITS1-F_KYO1, ITS1-F_KYO2, ITS2_KYO2, ITS3_KYO2, and ITS4 matched with 96.0–99.2% of the sequences examined in silico.”
Towards next‐generation biodiversity assessment using DNA metabarcoding (2012, methodology): “Several bioinformatic tools have recently been designed for finding the most optimal metabarcodes and primers (ecoPrimers program; Riaz et al. 2011) and for testing their characteristics (ecoPCR program; Bellemain et al. 2010; Ficetola et al. 2010).”
ecoPrimers: inference of new DNA barcode markers from whole genome sequence analysis (2011, background): “(11) to demonstrate the importance of primer selection for metabarcoding studies of fungal communities.”
Environmental DNA - An emerging tool in conservation for monitoring past and present biodiversity (2015, background): “…COI for animals (Hebert et al., 2003), the plastid ribulose 1,5- bisphosphate carboxylase gene (rbcL) and the maturase K gene (matK) for plants (Hollingsworth et al., 2009) and the internal transcribed spacer (ITS) for fungi (Nilsson et al., 2009; Bellemain et al., 2010; Schoch et al., 2012).”