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

Bellemain E, Carlsen T, Brochmann C, Coissac E, Taberlet P, Kauserud H - BMC Microbiol. (2010)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Centre for Biosystematics, University of Oslo, Norway. eva.bellemain@nhm.uio.no

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

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Commonly used primers for amplifying parts or the entirety of the ITS region. a) Relative position of the primers, design of the subsets and number of sequences in each subset. b) Primer sequences, references and position of the primer sequence according to a reference sequence of Serpula himantioides (AM946630) stretching the entire nrDNA repeat.
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Figure 1: Commonly used primers for amplifying parts or the entirety of the ITS region. a) Relative position of the primers, design of the subsets and number of sequences in each subset. b) Primer sequences, references and position of the primer sequence according to a reference sequence of Serpula himantioides (AM946630) stretching the entire nrDNA repeat.

Mentions: The ITS region includes the ITS1 and ITS2 regions, separated by the 5.8S gene, and is situated between the 18S (SSU) and 28S (LSU) genes in the nrDNA repeat unit (Figure 1). The large number of ITS copies per cell (up to 250; [13]) makes the region an appealing target for sequencing environmental substrates where the quantity of DNA present is low. The entire ITS region has commonly been targeted with traditional Sanger sequencing approaches and typically ranges between 450 and 700 bp. Either the ITS1 or the ITS2 region have been targeted in recent high-throughput sequencing studies [14-17], because the entire ITS region is still too long for 454 sequencing or other high-throughput sequencing methods. Using high-throughput sequencing, thousands of sequences can be analysed from a single environmental sample, enabling in-depth analysis of the fungal diversity. Various primers are used for amplifying the entire or parts of the ITS region (Figure 1). The most commonly used primers were published early in the 1990's (e.g. [18,19] when only a small fraction of the molecular variation in the nrDNA repeat across the fungal kingdom was known. Several other ITS primers have been published more recently [20] but have not been used extensively compared to the earlier published primers. However, little is actually known about the potential biases that commonly used ITS primers introduce during PCR amplification. Especially during high-throughput sequencing, where quantification (or semi-quantification) of species abundances is also possible to a certain degree (although hampered by factors like copy-number variation), primer mismatches might potentially introduce large biases in the results because some taxonomic groups are favoured during PCR. Our main focus in this study is on the two dominating taxonomic groups of fungi in the Dikarya, Ascomycota and Basidiomycota. Ascomycota represents the largest phylum of Fungi, with over 64,000 species, while Basidiomycota contains about 30,000 described species [21]. In total those two groups represent 79% of the described species of true Fungi.


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

Bellemain E, Carlsen T, Brochmann C, Coissac E, Taberlet P, Kauserud H - BMC Microbiol. (2010)

Commonly used primers for amplifying parts or the entirety of the ITS region. a) Relative position of the primers, design of the subsets and number of sequences in each subset. b) Primer sequences, references and position of the primer sequence according to a reference sequence of Serpula himantioides (AM946630) stretching the entire nrDNA repeat.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2909996&req=5

Figure 1: Commonly used primers for amplifying parts or the entirety of the ITS region. a) Relative position of the primers, design of the subsets and number of sequences in each subset. b) Primer sequences, references and position of the primer sequence according to a reference sequence of Serpula himantioides (AM946630) stretching the entire nrDNA repeat.
Mentions: The ITS region includes the ITS1 and ITS2 regions, separated by the 5.8S gene, and is situated between the 18S (SSU) and 28S (LSU) genes in the nrDNA repeat unit (Figure 1). The large number of ITS copies per cell (up to 250; [13]) makes the region an appealing target for sequencing environmental substrates where the quantity of DNA present is low. The entire ITS region has commonly been targeted with traditional Sanger sequencing approaches and typically ranges between 450 and 700 bp. Either the ITS1 or the ITS2 region have been targeted in recent high-throughput sequencing studies [14-17], because the entire ITS region is still too long for 454 sequencing or other high-throughput sequencing methods. Using high-throughput sequencing, thousands of sequences can be analysed from a single environmental sample, enabling in-depth analysis of the fungal diversity. Various primers are used for amplifying the entire or parts of the ITS region (Figure 1). The most commonly used primers were published early in the 1990's (e.g. [18,19] when only a small fraction of the molecular variation in the nrDNA repeat across the fungal kingdom was known. Several other ITS primers have been published more recently [20] but have not been used extensively compared to the earlier published primers. However, little is actually known about the potential biases that commonly used ITS primers introduce during PCR amplification. Especially during high-throughput sequencing, where quantification (or semi-quantification) of species abundances is also possible to a certain degree (although hampered by factors like copy-number variation), primer mismatches might potentially introduce large biases in the results because some taxonomic groups are favoured during PCR. Our main focus in this study is on the two dominating taxonomic groups of fungi in the Dikarya, Ascomycota and Basidiomycota. Ascomycota represents the largest phylum of Fungi, with over 64,000 species, while Basidiomycota contains about 30,000 described species [21]. In total those two groups represent 79% of the described species of true Fungi.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Centre for Biosystematics, University of Oslo, Norway. eva.bellemain@nhm.uio.no

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

Show MeSH
Related in: MedlinePlus