ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases
© Bellemain et al; licensee BioMed Central Ltd. 2010
Received: 9 April 2010
Accepted: 9 July 2010
Published: 9 July 2010
During 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.
Some 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.
We 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.
Molecular identification through DNA barcoding of fungi has, during the last 15-20 years, become an integrated and essential part of fungal ecology research and has provided new insights into the diversity and ecology of many different groups of fungi (reviewed by [1–4]). Molecular identification has made it possible to study the ecology of fungi in their dominant but inconspicuous mycelial stage and not only by means of fruiting bodies. Interest in sequenced-based analysis of environmental samples ('environmental barcoding') has increased in the past decade as it allows to study abundance and species richness of fungi at a high rate and more reliably than conventional biotic surveys (e.g. [5–10]). The internal transcribed spacer (ITS) of nuclear DNA (nrDNA) is the preferred DNA barcoding marker both for the identification of single taxa and mixed environmental templates ('environmental DNA barcoding'). It has recently been proposed as the official primary barcoding marker for fungi (Deliberation of 37 mycologists from 12 countries at the Smithsonian's Conservation and Research Centre, Front Royal, Virginia, May 2007). More than 100 000 fungal ITS sequences generated by conventional Sanger sequencing are deposited in the International Nucleotide Sequence Databases and/or other databases , providing a large reference material for identification of fungal taxa. However, these data are to some extent hampered by misidentifications or technical errors such as mixing of DNA templates or sequencing errors . Furthermore, a large amount of partial ITS sequences generated by next-generation sequencing has recently been deposited in public sequence databases.
The aim of this study was to analyse the biases commonly used ITS primers might introduce during PCR amplification. First, we addressed to what degree the various primers mismatch with the target sequence and whether the mismatches are more widespread in some taxonomic groups. Second, we considered the length variation in the amplified products, in relation to taxonomic group, to assess amplification biases during real (in vitro) PCR amplification, as shorter DNA fragments are preferentially amplified from environmental samples containing DNA from a mixture of different species . Finally, we analyzed to what degree the various primers co-amplify plants, which often co-occur in environmental samples. For these purposes we performed in silico PCR using various primer combinations on target sequences retrieved from EMBL databases as well as subset databases using the bioinformatic tool EcoPCR . In order to better simulate real PCR conditions, we allowed a maximum of 0 to 3 mismatches except for the 2 last bases of each primer and we assessed the melting temperature (Tm) for each primer in relation to primer mismatches.
Compilation of datasets
The EcoPCR package contains a set of bioinformatics tools developed at the Laboratoire d'Ecologie Alpine, Grenoble, France (, freely available at http://www.grenoble.prabi.fr/trac/ecoPCR). The package is composed of four pieces of software, namely 'ecoPCRFormat', 'ecoFind', 'ecoPCR' and 'ecoGrep'. Briefly, EcoPCR is based on the pattern matching algorithm agrep  and selects sequences from a database that match (exhibit similarity to) two PCR primers. The user can specify (1) which database the given primers should be tested against, and (2) the primer sequences. Different options allow specification of the minimum and maximum amplification length, the maximum count of mismatched positions between each primer and the target sequence (excluding the two bases on the 3'end of each primer), and restriction of the search to given taxonomic groups. The ecoPCR output contains, for each target sequence, amplification length, melting temperature (Tm), taxonomic information as well as the number of mismatched positions for each strand.
First, we retrieved from EMBL sequences from fungi in the following categories: 'standard', 'Genome sequence scan', 'High Throughput Genome sequencing', 'Whole Genome Sequence' from ftp://ftp.ebi.ac.uk/pub/databases/embl/release/ (release embl_102, January 2010) to create our initial database. It corresponds to 1,212,954 sequences including approximately 79,500 ITS sequences (estimated from EMBL SRS website requesting for fungi sequences annotated with 'ITS' or 'Internal Transcribed Spacer'). These ITS entries refer to more than 10,800 taxa. This database hereafter referred to as the "fungi database" was compiled using EcoPCRFormat.
To assess the specificity of the primers to fungi, we used the plant database from EMBL (release embl_102, January 2010 from ftp://ftp.ebi.ac.uk/pub/databases/embl/release/) to run amplifications using the same primers as for fungi. This database, hereafter referred to as the "plant database", contained 1,253,565 sequences, including approximately 65,000 ITS sequences (estimated from EMBL SRS website requesting for viridiplantae sequences annotated with 'ITS' or 'Internal Transcribed Spacer'). These ITS entries refer to more than 6,100 taxa. This database was also compiled using EcoPCRFormat.
As there are relatively few sequences submitted to public databases covering the entire ITS region as well as the commonly used universal primer sites in the flanking SSU and LSU regions, we created three subset datasets covering either ITS1, ITS2 or the entire ITS region. From the initial fungi database, we compiled three subset databases (hereafter referred to as subset 1, 2, and 3) by in silico amplification (see below) of target sequences using the following primer pairs: NS7-ITS2 (dataset 1, focused on ITS1 region), ITS5-ITS4 (dataset 2, including both ITS1 and ITS2 regions) and ITS3-LR3 (dataset 3, focused on ITS2 region). To simulate relatively stringent PCR conditions, a single mismatch between each primer and the template was allowed except in the 2 bases of the 3' primer end. These three subsets were then compiled using EcoPCRFormat and included 1291, 5924 and 2459 partial nrDNA sequences, respectively.
In silico amplification and primer specificity to fungi
Using EcoPCR, we ran in silico amplifications from both the fungi and the plant databases using various commonly used primer combinations, to assess the number of amplifications and the specificity of the primers to fungi. For each amplification, we allowed from 0 to 3 mismatches between each primer and the template (excluding mismatches in the 2 bases of the 3' primer end) in order to simulate different stringency conditions of PCRs. Secondly, from the three subsets, we amplified sequences using different internal primer combinations in order to evaluate the various primers (Figure 1). From dataset 1 we used the primer combinations ITS1-F-ITS2, ITS5-ITS2 and ITS1-ITS2. From dataset 2 we used the combinations ITS1-ITS4 (amplifying both ITS1 and ITS2 introns), ITS3-ITS4 and ITS5-ITS2. From dataset 3 we used the combinations ITS3-ITS4 and ITS3-ITS4B. During these virtual PCRs we also allowed from 0 to 3 mismatches between each primer and the template, except in the 2 bases of the 3' primer end.
Assessing the degree of primer mismatches and Tm
For all in silico internal amplifications from each subset, we assessed the proportion of sequences retrieved when allowing for 0 to 3 mismatches between each primer and the template. For the amplifications from each subset, we used an external primer (one of the primers used to create the subset) and an internal primer. Therefore, for each analysis, we assessed the proportion of sequences including mismatches for the internal primer only. The primer pair ITS5-ITS2 was evaluated both for subset 1 and subset 2, with the focus on ITS5 for subset 1 and on ITS2 for subset 2 (as those primers correspond to internal primers within their respective subsets). Similarly, the primer pair ITS3-ITS4 was evaluated both for subsets 2 and 3, with the focus on ITS3 in subset 2 and ITS4 in subset 3. The primer ITS1 was evaluated both for subset 1 (with the combination ITS1-ITS2) and for subset 2 (with the combination ITS1-ITS4) as ITS2 and ITS4 were used as external primers in subsets 1 and 2, respectively.
To assess whether certain taxonomic groups were more prone to mismatches, we assessed the proportion of sequences including one mismatch for each of the three taxonomic groups 'ascomycetes', 'basidiomycetes' and 'non-dikarya' (the latter is a highly polyphyletic group including e.g. Blastocladiomycota, Chytridiomycota, Glomeromycota and Zygomycota ). We also assessed the Tm for each primer based on the analyses from internal amplifications, allowing a single mismatch. The Tm is defined as the temperature at which half of the DNA strands are in the double-helical state and half are in the "random-coil" states. The strength of hybridization between the primers and the template affects Tm. It is therefore informative to assess how Tm decreases as the number of mismatches increases, i.e. with less stringent PCR conditions. Tm was calculated in ecoPCR based on a thermodynamic nearest neighbor model . Exact computation was performed following .
Assessing bias in amplification length relative to taxonomic group
To further assess the taxonomic bias introduced by the use of the different primer pairs, we separated the amplified sequences from selected analyses into the groups 'ascomycetes', 'basidomycetes' and 'non-dikarya' based on their taxonomic identification number, using the ecoGrep tool. These selected analyses were (1) the three subsets, and (2) all internal amplifications within each subset with one mismatch allowed. The amplification length was reported for each analysis.
Relative amplification of different primer combinations from the fungi and plant databases
Number of plant and fungi ITS sequences amplified in silico from EMBL fungal and plant databases, using the various primer combinations and allowing none to three mismatches.
Fungal ITS sequences
Plant ITS sequences
Number of mismatches *
Primer mismatches in sequence subsets
Allowing one mismatch increased the proportion of amplified sequences from 36% to 91.6% for the commonly used primer ITS1-F, implying that more than half of the amplified sequences included one mismatch. ITS5 amplified the highest proportion of the sequences when allowing for a single mismatch (97.5%), and less than 10% of the sequences in each taxonomic group included one mismatch. The primer ITS1, on the other hand, only amplified 56.8% and 65.9% of the sequences from subsets one and two, respectively, when allowing no mismatches. Allowing three mismatches, ITS1 was still only able to amplify 92% of the sequences in subsets one and two. Allowing no mismatches, the complementary primers ITS2 and ITS3 amplified 79.4% and 77.3% of all sequences respectively, in subset 2. Allowing one mismatch, these numbers increased to 87.5 and 90%, respectively. Primer ITS4 amplified 74.9% of all sequences in subset 3 and this proportion only increased to 93.7% when allowing three mismatches. The assumed basidiomycete-specific primer ITS4-B amplified only 5.6% of the sequences in subset 3 under strict conditions (corresponding to 46% of the basidiomycetes sequences, see below) and up to 14.9% allowing 3 mismatches. However, about half of the sequences included a mismatch when a single mismatch was allowed.
Taxonomic bias for different primers
Percentage of sequences amplified in silico, allowing one mismatch, from ascomycetes, basidiomycetes and 'non-Dikarya' with different primer combinations and using the three sequence subsets 1-3 (see Material and Methods) as templates.
Melting temperature (Tm) of each primer according to the number of mismatches allowed between the primer and the target sequence.
Number of mismatches allowed
Taxonomic bias relative to length of the amplified region
Number (percentages) of sequences and amplified in each of the most common Basidiomycete groups, from the original subset3 and from the amplification of ITS3-ITS4-B from subset3, allowing no or 3 mismatches.
Other categories *
Our in silico analyses further indicate that most of the primers will introduce a taxonomic bias due to higher levels of mismatches in certain taxonomic groups. When allowing one mismatch (corresponding to rather stringent PCR conditions) we found that the primer pairs ITS1-F, ITS1 and ITS5 preferentially amplified basidiomycetes whereas the primer pairs ITS2, ITS3 and ITS4 preferentially amplified ascomycetes. This type of bias must also be considered before selecting primer pairs for a given study. Also in molecular surveys of protistan and prokaryotic diversity, it has been documented that different 16S primers target different parts of the diversity [32–34].
In addition, our results clearly demonstrate that basidiomycetes, on average, have significantly longer amplicon sequences than ascomycetes both for the whole ITS region, and the ITS2 region. This fact probably also introduces taxonomic bias during PCR amplification of environmental samples, since shorter fragments are more readily amplified compared to longer ones. In several studies, it has been demonstrated that a greater proportion of the diversity can be detected with short target sequences compared to longer ones [35, 36]. Hence, using the ITS2 region or the whole ITS region, a higher number of the ascomycetes will probably be targeted compared to basidiomycetes. This bias could be avoided by using primers amplifying ITS1 only, but this would imply a preferential amplification of the 'non-dikarya' fungi.
The in silico method used here allowed for the assessment of different parameters for commonly used ITS primers, including the length amplicons generated, taxonomic biases, and the consequences of primer mismatches. The results provide novel insights into the relative performance of commonly used ITS primer pairs. Our analyses suggest that studies using these ITS primers to retrieve the entire fungal diversity from environmental samples including mixed templates should use lower annealing temperatures than the recommended Tm to allow for primer mismatches. A high Tm has been used in most studies, which likely biases the inferred taxonomic composition and diversity. However, one has to find a balance between allowing some mismatches and avoiding non-specific binding in other genomic regions, which can also be a problem.
Considering the different types of biases (specificity to fungi; mismatches; length; taxonomy), we suggest that different primer combinations targeting different parts of the ITS region should be analyzed in parallel. When dealing with single culture isolates compared to environmental samples, the choice of a primer pair to amplify ITS is less problematic because there is no 'competition' between DNA fragments of different taxonomic groups/lengths, and the DNA quality is generally higher.
This study also illustrates potential benefits of using a bioinformatics approach before selecting primer pairs for a given study. We nevertheless emphasize that an in silico analysis does not necessarily reflect the performance of the primers in vitro, since there are many other PCR parameters such as ITS copy number, amplification program, and salt and primer concentration in the PCR mix that cannot easily be simulated. This study should therefore be followed up by in vitro PCR analyses of the fungal ITS primers where biases are measured based on sequence output, although it will be a huge task to control and check for all types of biases that might be involved. We are currently performing further bioinformatics analyses using the tool 'ecoPrimer' (http://www.grenoble.prabi.fr/trac/ecoPrimers; Riaz et al. unpublished) to identify the most appropriate barcoding primers within the ITS region and other regions, with the intent of determining whether new ITS primers, such as those recently published by Martin and Rygiewicz , should replace the currently used ones.
Eva Bellemain was funded by the Natural History Museum, University of Oslo and this work has been initiated as part of the BarFrost project (Barcoding of permafrost samples). We are thankful to four anonymous reviewers for constructive comments and to Marie Davey for helping to improve the style of written English.
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