Comparison and Validation of Some ITS Primer Pairs Useful for Fungal Metabarcoding Studies
Identifieur interne : 000102 ( Pmc/Checkpoint ); précédent : 000101; suivant : 000103Comparison and Validation of Some ITS Primer Pairs Useful for Fungal Metabarcoding Studies
Auteurs : Michiel Op De Beeck [Belgique] ; Bart Lievens [Belgique] ; Pieter Busschaert [Belgique] ; Stéphan Declerck [Belgique] ; Jaco Vangronsveld [Belgique] ; Jan V. Colpaert [Belgique]Source :
- PLoS ONE [ 1932-6203 ] ; 2014.
Abstract
Current metabarcoding studies aiming to characterize microbial communities generally rely on the amplification and sequencing of relatively short DNA regions. For fungi, the internal transcribed spacer (ITS) region in the ribosomal RNA (rRNA) operon has been accepted as the formal fungal barcode. Despite an increasing number of fungal metabarcoding studies, the amplification efficiency of primers is generally not tested prior to their application in metabarcoding studies. Some of the challenges that metabarcoding primers should overcome efficiently are the amplification of target DNA strands in samples rich in non-target DNA and environmental pollutants, such as humic acids, that may have been co-extracted with DNA. In the current study, three selected primer pairs were tested for their suitability as fungal metabarcoding primers. The selected primer pairs include two primer pairs that have been frequently used in fungal metabarcoding studies (ITS1F/ITS2 and ITS3/ITS4) and a primer pair (ITS86F/ITS4) that has been shown to efficiently amplify the ITS2 region of a broad range of fungal taxa in environmental soil samples. The selected primer pairs were evaluated in a 454 amplicon pyrosequencing experiment, real-time PCR (qPCR) experiments and
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DOI: 10.1371/journal.pone.0097629
PubMed: 24933453
PubMed Central: 4059633
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Comparison and Validation of Some ITS Primer Pairs Useful for Fungal Metabarcoding Studies</title><author><name sortKey="Op De Beeck, Michiel" sort="Op De Beeck, Michiel" uniqKey="Op De Beeck M" first="Michiel" last="Op De Beeck">Michiel Op De Beeck</name><affiliation wicri:level="1"><nlm:aff id="aff1"><addr-line>Centre for Environmental Sciences, Hasselt University, Hasselt, Limburg, Belgium</addr-line></nlm:aff><country xml:lang="fr">Belgique</country><wicri:regionArea>Centre for Environmental Sciences, Hasselt University, Hasselt, Limburg</wicri:regionArea><wicri:noRegion>Limburg</wicri:noRegion></affiliation></author><author><name sortKey="Lievens, Bart" sort="Lievens, Bart" uniqKey="Lievens B" first="Bart" last="Lievens">Bart Lievens</name><affiliation wicri:level="1"><nlm:aff id="aff2"><addr-line>Department of Microbial and Molecular Systems (M2S), Catholic University of Leuven, Antwerp, Belgium</addr-line></nlm:aff><country xml:lang="fr">Belgique</country><wicri:regionArea>Department of Microbial and Molecular Systems (M2S), Catholic University of Leuven, Antwerp</wicri:regionArea><wicri:noRegion>Antwerp</wicri:noRegion></affiliation></author><author><name sortKey="Busschaert, Pieter" sort="Busschaert, Pieter" uniqKey="Busschaert P" first="Pieter" last="Busschaert">Pieter Busschaert</name><affiliation wicri:level="1"><nlm:aff id="aff2"><addr-line>Department of Microbial and Molecular Systems (M2S), Catholic University of Leuven, Antwerp, Belgium</addr-line></nlm:aff><country xml:lang="fr">Belgique</country><wicri:regionArea>Department of Microbial and Molecular Systems (M2S), Catholic University of Leuven, Antwerp</wicri:regionArea><wicri:noRegion>Antwerp</wicri:noRegion></affiliation></author><author><name sortKey="Declerck, Stephan" sort="Declerck, Stephan" uniqKey="Declerck S" first="Stéphan" last="Declerck">Stéphan Declerck</name><affiliation wicri:level="1"><nlm:aff id="aff3"><addr-line>Earth & Life Institute, Catholic University of Louvain, Louvain-la-Neuve, Walloon Brabant, Belgium</addr-line></nlm:aff><country xml:lang="fr">Belgique</country><wicri:regionArea>Earth & Life Institute, Catholic University of Louvain, Louvain-la-Neuve, Walloon Brabant</wicri:regionArea><wicri:noRegion>Walloon Brabant</wicri:noRegion></affiliation></author><author><name sortKey="Vangronsveld, Jaco" sort="Vangronsveld, Jaco" uniqKey="Vangronsveld J" first="Jaco" last="Vangronsveld">Jaco Vangronsveld</name><affiliation wicri:level="1"><nlm:aff id="aff1"><addr-line>Centre for Environmental Sciences, Hasselt University, Hasselt, Limburg, Belgium</addr-line></nlm:aff><country xml:lang="fr">Belgique</country><wicri:regionArea>Centre for Environmental Sciences, Hasselt University, Hasselt, Limburg</wicri:regionArea><wicri:noRegion>Limburg</wicri:noRegion></affiliation></author><author><name sortKey="Colpaert, Jan V" sort="Colpaert, Jan V" uniqKey="Colpaert J" first="Jan V." last="Colpaert">Jan V. 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Colpaert</name><affiliation wicri:level="1"><nlm:aff id="aff1"><addr-line>Centre for Environmental Sciences, Hasselt University, Hasselt, Limburg, Belgium</addr-line></nlm:aff><country xml:lang="fr">Belgique</country><wicri:regionArea>Centre for Environmental Sciences, Hasselt University, Hasselt, Limburg</wicri:regionArea><wicri:noRegion>Limburg</wicri:noRegion></affiliation></author></analytic><series><title level="j">PLoS ONE</title><idno type="eISSN">1932-6203</idno><imprint><date when="2014">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Current metabarcoding studies aiming to characterize microbial communities generally rely on the amplification and sequencing of relatively short DNA regions. For fungi, the internal transcribed spacer (ITS) region in the ribosomal RNA (rRNA) operon has been accepted as the formal fungal barcode. Despite an increasing number of fungal metabarcoding studies, the amplification efficiency of primers is generally not tested prior to their application in metabarcoding studies. Some of the challenges that metabarcoding primers should overcome efficiently are the amplification of target DNA strands in samples rich in non-target DNA and environmental pollutants, such as humic acids, that may have been co-extracted with DNA. In the current study, three selected primer pairs were tested for their suitability as fungal metabarcoding primers. The selected primer pairs include two primer pairs that have been frequently used in fungal metabarcoding studies (ITS1F/ITS2 and ITS3/ITS4) and a primer pair (ITS86F/ITS4) that has been shown to efficiently amplify the ITS2 region of a broad range of fungal taxa in environmental soil samples. The selected primer pairs were evaluated in a 454 amplicon pyrosequencing experiment, real-time PCR (qPCR) experiments and <italic>in silico</italic> analyses. Results indicate that experimental evaluation of primers provides valuable information that could aid in the selection of suitable primers for fungal metabarcoding studies. Furthermore, we show that the ITS86F/ITS4 primer pair outperforms other primer pairs tested in terms of <italic>in silico</italic> primer efficiency, PCR efficiency, coverage, number of reads and number of species-level operational taxonomic units (OTUs) obtained. These traits push the ITS86F/ITS4 primer pair forward as highly suitable for studying fungal diversity and community structures using DNA metabarcoding.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Stahl, Da" uniqKey="Stahl D">DA Stahl</name></author><author><name sortKey="Lane, Dj" uniqKey="Lane D">DJ Lane</name></author><author><name sortKey="Olsen, Gj" uniqKey="Olsen G">GJ Olsen</name></author><author><name sortKey="Pace, Nr" uniqKey="Pace N">NR Pace</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hugenholtz, P" uniqKey="Hugenholtz P">P Hugenholtz</name></author><author><name sortKey="Pace, Nr" uniqKey="Pace N">NR Pace</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Margulies, M" uniqKey="Margulies M">M Margulies</name></author><author><name sortKey="Egholm, M" uniqKey="Egholm M">M Egholm</name></author><author><name sortKey="Altman, We" uniqKey="Altman 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<front><journal-meta><journal-id journal-id-type="nlm-ta">PLoS One</journal-id><journal-id journal-id-type="iso-abbrev">PLoS ONE</journal-id><journal-id journal-id-type="publisher-id">plos</journal-id><journal-id journal-id-type="pmc">plosone</journal-id><journal-title-group><journal-title>PLoS ONE</journal-title></journal-title-group><issn pub-type="epub">1932-6203</issn><publisher><publisher-name>Public Library of Science</publisher-name><publisher-loc>San Francisco, USA</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">24933453</article-id><article-id pub-id-type="pmc">4059633</article-id><article-id pub-id-type="publisher-id">PONE-D-14-00713</article-id><article-id pub-id-type="doi">10.1371/journal.pone.0097629</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group><subj-group subj-group-type="Discipline-v2"><subject>Biology and Life Sciences</subject><subj-group><subject>Ecology</subject><subj-group><subject>Plant Ecology</subject><subj-group><subject>Plant-Environment Interactions</subject></subj-group></subj-group><subj-group><subject>Biodiversity</subject><subject>Community Ecology</subject><subject>Microbial Ecology</subject></subj-group></subj-group><subj-group><subject>Microbiology</subject><subj-group><subject>Plant Microbiology</subject></subj-group></subj-group><subj-group><subject>Mycology</subject></subj-group><subj-group><subject>Organisms</subject><subj-group><subject>Fungi</subject></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v2"><subject>Ecology and Environmental Sciences</subject><subj-group><subject>Soil Science</subject><subj-group><subject>Soil Ecology</subject></subj-group></subj-group></subj-group></article-categories><title-group><article-title>Comparison and Validation of Some ITS Primer Pairs Useful for Fungal Metabarcoding Studies</article-title><alt-title alt-title-type="running-head">Primers Used in Fungal Metabarcoding Studies</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Op De Beeck</surname><given-names>Michiel</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Lievens</surname><given-names>Bart</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Busschaert</surname><given-names>Pieter</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Declerck</surname><given-names>Stéphan</given-names></name><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Vangronsveld</surname><given-names>Jaco</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Colpaert</surname><given-names>Jan V.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib></contrib-group><aff id="aff1"><label>1</label><addr-line>Centre for Environmental Sciences, Hasselt University, Hasselt, Limburg, Belgium</addr-line></aff><aff id="aff2"><label>2</label><addr-line>Department of Microbial and Molecular Systems (M2S), Catholic University of Leuven, Antwerp, Belgium</addr-line></aff><aff id="aff3"><label>3</label><addr-line>Earth & Life Institute, Catholic University of Louvain, Louvain-la-Neuve, Walloon Brabant, Belgium</addr-line></aff><contrib-group><contrib contrib-type="editor"><name><surname>Neilan</surname><given-names>Brett</given-names></name><role>Editor</role><xref ref-type="aff" rid="edit1"></xref></contrib></contrib-group><aff id="edit1"><addr-line>University of New South Wales, Australia</addr-line></aff><author-notes><corresp id="cor1">* E-mail: <email>jan.colpaert@uhasselt.be</email></corresp><fn fn-type="conflict"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type="con"><p>Conceived and designed the experiments: MODB JV JVC. Performed the experiments: MODB. Analyzed the data: MODB PB. Contributed reagents/materials/analysis tools: PB BL SD. Wrote the paper: MODB.</p></fn></author-notes><pub-date pub-type="collection"><year>2014</year></pub-date><pub-date pub-type="epub"><day>16</day><month>6</month><year>2014</year></pub-date><volume>9</volume><issue>6</issue><elocation-id>e97629</elocation-id><history><date date-type="received"><day>17</day><month>1</month><year>2014</year></date><date date-type="accepted"><day>21</day><month>4</month><year>2014</year></date></history><permissions><copyright-year>2014</copyright-year><copyright-holder>Op De Beeck et al</copyright-holder><license><license-p>This is an open-access article distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><abstract><p>Current metabarcoding studies aiming to characterize microbial communities generally rely on the amplification and sequencing of relatively short DNA regions. For fungi, the internal transcribed spacer (ITS) region in the ribosomal RNA (rRNA) operon has been accepted as the formal fungal barcode. Despite an increasing number of fungal metabarcoding studies, the amplification efficiency of primers is generally not tested prior to their application in metabarcoding studies. Some of the challenges that metabarcoding primers should overcome efficiently are the amplification of target DNA strands in samples rich in non-target DNA and environmental pollutants, such as humic acids, that may have been co-extracted with DNA. In the current study, three selected primer pairs were tested for their suitability as fungal metabarcoding primers. The selected primer pairs include two primer pairs that have been frequently used in fungal metabarcoding studies (ITS1F/ITS2 and ITS3/ITS4) and a primer pair (ITS86F/ITS4) that has been shown to efficiently amplify the ITS2 region of a broad range of fungal taxa in environmental soil samples. The selected primer pairs were evaluated in a 454 amplicon pyrosequencing experiment, real-time PCR (qPCR) experiments and <italic>in silico</italic> analyses. Results indicate that experimental evaluation of primers provides valuable information that could aid in the selection of suitable primers for fungal metabarcoding studies. Furthermore, we show that the ITS86F/ITS4 primer pair outperforms other primer pairs tested in terms of <italic>in silico</italic> primer efficiency, PCR efficiency, coverage, number of reads and number of species-level operational taxonomic units (OTUs) obtained. These traits push the ITS86F/ITS4 primer pair forward as highly suitable for studying fungal diversity and community structures using DNA metabarcoding.</p></abstract><funding-group><funding-statement>The study was made possible by funding of the Research Foundation Flanders (FWO). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.</funding-statement></funding-group><counts><page-count count="11"></page-count></counts></article-meta></front></pmc><affiliations><list><country><li>Belgique</li></country></list><tree><country name="Belgique"><noRegion><name sortKey="Op De Beeck, Michiel" sort="Op De Beeck, Michiel" uniqKey="Op De Beeck M" first="Michiel" last="Op De Beeck">Michiel Op De Beeck</name></noRegion><name sortKey="Busschaert, Pieter" sort="Busschaert, Pieter" uniqKey="Busschaert P" first="Pieter" last="Busschaert">Pieter Busschaert</name><name sortKey="Colpaert, Jan V" sort="Colpaert, Jan V" uniqKey="Colpaert J" first="Jan V." last="Colpaert">Jan V. Colpaert</name><name sortKey="Declerck, Stephan" sort="Declerck, Stephan" uniqKey="Declerck S" first="Stéphan" last="Declerck">Stéphan Declerck</name><name sortKey="Lievens, Bart" sort="Lievens, Bart" uniqKey="Lievens B" first="Bart" last="Lievens">Bart Lievens</name><name sortKey="Vangronsveld, Jaco" sort="Vangronsveld, Jaco" uniqKey="Vangronsveld J" first="Jaco" last="Vangronsveld">Jaco Vangronsveld</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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