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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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Related in: MedlinePlus

Percentage of sequences amplified from each subset using different primer pairs allowing a maximum of 0, 1, 2, or 3 mismatches.
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Figure 2: Percentage of sequences amplified from each subset using different primer pairs allowing a maximum of 0, 1, 2, or 3 mismatches.

Mentions: The selected ITS primers showed large variation in their ability to amplify fungal sequences from the three subsets when allowing different number of mismatches (Figure 2). All primer pairs amplified at least 90% of the sequences when allowing two or three mismatches, with the exception of ITS4-B (see below). It is noteworthy that the percentages of sequences were quite similar for two and three mismatches, indicating that rather few sequences included three mismatches. Under strict conditions (i.e. allowing no mismatches), the proportion of amplified sequences varied considerably between primer pairs, ranging from 36% for ITS1-F to 81% for ITS5 (Figure 2).


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)

Percentage of sequences amplified from each subset using different primer pairs allowing a maximum of 0, 1, 2, or 3 mismatches.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Percentage of sequences amplified from each subset using different primer pairs allowing a maximum of 0, 1, 2, or 3 mismatches.
Mentions: The selected ITS primers showed large variation in their ability to amplify fungal sequences from the three subsets when allowing different number of mismatches (Figure 2). All primer pairs amplified at least 90% of the sequences when allowing two or three mismatches, with the exception of ITS4-B (see below). It is noteworthy that the percentages of sequences were quite similar for two and three mismatches, indicating that rather few sequences included three mismatches. Under strict conditions (i.e. allowing no mismatches), the proportion of amplified sequences varied considerably between primer pairs, ranging from 36% for ITS1-F to 81% for ITS5 (Figure 2).

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