Performance of 16s rDNA Primer Pairs in the Study of Rhizosphere and Endosphere Bacterial Microbiomes in Metabarcoding Studies.
Identifieur interne : 001734 ( Main/Exploration ); précédent : 001733; suivant : 001735Performance of 16s rDNA Primer Pairs in the Study of Rhizosphere and Endosphere Bacterial Microbiomes in Metabarcoding Studies.
Auteurs : Bram Beckers [Belgique] ; Michiel Op De Beeck [Belgique] ; Sofie Thijs [Belgique] ; Sascha Truyens [Belgique] ; Nele Weyens [Belgique] ; Wout Boerjan [Belgique] ; Jaco Vangronsveld [Belgique]Source :
- Frontiers in microbiology [ 1664-302X ] ; 2016.
Abstract
Next-generation sequencing technologies have revolutionized the methods for studying microbial ecology by enabling high-resolution community profiling. However, the use of these technologies in unraveling the plant microbiome remains challenging. Many bacterial 16S rDNA primer pairs also exhibit high affinity for non-target DNA such as plastid (mostly chloroplast) DNA and mitochondrial DNA. Therefore, we experimentally tested a series of commonly used primers for the analysis of plant-associated bacterial communities using 454 pyrosequencing. We evaluated the performance of all selected primer pairs in the study of the bacterial microbiomes present in the rhizosphere soil, root, stem and leaf endosphere of field-grown poplar trees (Populus tremula × Populus alba) based on (a) co-amplification of non-target DNA, (b) low amplification efficiency for pure chloroplast DNA (real-time PCR), (c) high retrieval of bacterial 16S rDNA, (d) high operational taxonomic unit (OTU) richness and Inverse Simpson diversity and (e) taxonomic assignment of reads. Results indicate that experimental evaluation of primers provide valuable information that could contribute in the selection of suitable primer pairs for 16S rDNA metabarcoding studies in plant-microbiota research. Furthermore, we show that primer pair 799F-1391R outperforms all other primer pairs in our study in the elimination of non-target DNA and retrieval of bacterial OTUs.
DOI: 10.3389/fmicb.2016.00650
PubMed: 27242686
PubMed Central: PMC4865482
Affiliations:
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Department of Plant Biotechnology and Bioinformatics, Ghent UniversityGent, Belgium.</nlm:affiliation><country xml:lang="fr">Belgique</country><wicri:regionArea>Department of Plant Systems Biology, Flanders Institute for Biotechnology (VIB)Gent, Belgium; Department of Plant Biotechnology and Bioinformatics, Ghent UniversityGent</wicri:regionArea><wicri:noRegion>Ghent UniversityGent</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:affiliation>Centre for Environmental Sciences, Hasselt University Diepenbeek, Belgium.</nlm:affiliation><country xml:lang="fr">Belgique</country><wicri:regionArea>Centre for Environmental Sciences, Hasselt University Diepenbeek</wicri:regionArea><wicri:noRegion>Hasselt University Diepenbeek</wicri:noRegion></affiliation></author></analytic><series><title level="j">Frontiers in microbiology</title><idno type="ISSN">1664-302X</idno><imprint><date when="2016" type="published">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Next-generation sequencing technologies have revolutionized the methods for studying microbial ecology by enabling high-resolution community profiling. However, the use of these technologies in unraveling the plant microbiome remains challenging. Many bacterial 16S rDNA primer pairs also exhibit high affinity for non-target DNA such as plastid (mostly chloroplast) DNA and mitochondrial DNA. Therefore, we experimentally tested a series of commonly used primers for the analysis of plant-associated bacterial communities using 454 pyrosequencing. We evaluated the performance of all selected primer pairs in the study of the bacterial microbiomes present in the rhizosphere soil, root, stem and leaf endosphere of field-grown poplar trees (Populus tremula × Populus alba) based on (a) co-amplification of non-target DNA, (b) low amplification efficiency for pure chloroplast DNA (real-time PCR), (c) high retrieval of bacterial 16S rDNA, (d) high operational taxonomic unit (OTU) richness and Inverse Simpson diversity and (e) taxonomic assignment of reads. Results indicate that experimental evaluation of primers provide valuable information that could contribute in the selection of suitable primer pairs for 16S rDNA metabarcoding studies in plant-microbiota research. Furthermore, we show that primer pair 799F-1391R outperforms all other primer pairs in our study in the elimination of non-target DNA and retrieval of bacterial OTUs. </div></front></TEI><pubmed><MedlineCitation Status="PubMed-not-MEDLINE" Owner="NLM"><PMID Version="1">27242686</PMID><DateCompleted><Year>2016</Year><Month>05</Month><Day>31</Day></DateCompleted><DateRevised><Year>2020</Year><Month>09</Month><Day>29</Day></DateRevised><Article PubModel="Electronic-eCollection"><Journal><ISSN IssnType="Print">1664-302X</ISSN><JournalIssue CitedMedium="Print"><Volume>7</Volume><PubDate><Year>2016</Year></PubDate></JournalIssue><Title>Frontiers in microbiology</Title><ISOAbbreviation>Front Microbiol</ISOAbbreviation></Journal><ArticleTitle>Performance of 16s rDNA Primer Pairs in the Study of Rhizosphere and Endosphere Bacterial Microbiomes in Metabarcoding Studies.</ArticleTitle><Pagination><MedlinePgn>650</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.3389/fmicb.2016.00650</ELocationID><Abstract><AbstractText>Next-generation sequencing technologies have revolutionized the methods for studying microbial ecology by enabling high-resolution community profiling. However, the use of these technologies in unraveling the plant microbiome remains challenging. Many bacterial 16S rDNA primer pairs also exhibit high affinity for non-target DNA such as plastid (mostly chloroplast) DNA and mitochondrial DNA. Therefore, we experimentally tested a series of commonly used primers for the analysis of plant-associated bacterial communities using 454 pyrosequencing. We evaluated the performance of all selected primer pairs in the study of the bacterial microbiomes present in the rhizosphere soil, root, stem and leaf endosphere of field-grown poplar trees (Populus tremula × Populus alba) based on (a) co-amplification of non-target DNA, (b) low amplification efficiency for pure chloroplast DNA (real-time PCR), (c) high retrieval of bacterial 16S rDNA, (d) high operational taxonomic unit (OTU) richness and Inverse Simpson diversity and (e) taxonomic assignment of reads. Results indicate that experimental evaluation of primers provide valuable information that could contribute in the selection of suitable primer pairs for 16S rDNA metabarcoding studies in plant-microbiota research. Furthermore, we show that primer pair 799F-1391R outperforms all other primer pairs in our study in the elimination of non-target DNA and retrieval of bacterial OTUs. </AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Beckers</LastName><ForeName>Bram</ForeName><Initials>B</Initials><AffiliationInfo><Affiliation>Centre for Environmental Sciences, Hasselt University Diepenbeek, Belgium.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Op De Beeck</LastName><ForeName>Michiel</ForeName><Initials>M</Initials><AffiliationInfo><Affiliation>Centre for Environmental Sciences, Hasselt University Diepenbeek, Belgium.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Thijs</LastName><ForeName>Sofie</ForeName><Initials>S</Initials><AffiliationInfo><Affiliation>Centre for Environmental Sciences, Hasselt University Diepenbeek, Belgium.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Truyens</LastName><ForeName>Sascha</ForeName><Initials>S</Initials><AffiliationInfo><Affiliation>Centre for Environmental Sciences, Hasselt University Diepenbeek, Belgium.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Weyens</LastName><ForeName>Nele</ForeName><Initials>N</Initials><AffiliationInfo><Affiliation>Centre for Environmental Sciences, Hasselt University Diepenbeek, Belgium.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Boerjan</LastName><ForeName>Wout</ForeName><Initials>W</Initials><AffiliationInfo><Affiliation>Department of Plant Systems Biology, Flanders Institute for Biotechnology (VIB)Gent, Belgium; Department of Plant Biotechnology and Bioinformatics, Ghent UniversityGent, Belgium.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Vangronsveld</LastName><ForeName>Jaco</ForeName><Initials>J</Initials><AffiliationInfo><Affiliation>Centre for Environmental Sciences, Hasselt University Diepenbeek, Belgium.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2016</Year><Month>05</Month><Day>13</Day></ArticleDate></Article><MedlineJournalInfo><Country>Switzerland</Country><MedlineTA>Front Microbiol</MedlineTA><NlmUniqueID>101548977</NlmUniqueID><ISSNLinking>1664-302X</ISSNLinking></MedlineJournalInfo><KeywordList Owner="NOTNLM"><Keyword MajorTopicYN="N">16S rDNA metabarcoding</Keyword><Keyword MajorTopicYN="N">454 pyrosequencing</Keyword><Keyword MajorTopicYN="N">chloroplast DNA</Keyword><Keyword MajorTopicYN="N">endophytes</Keyword><Keyword MajorTopicYN="N">plant microbiome</Keyword></KeywordList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="received"><Year>2015</Year><Month>11</Month><Day>12</Day></PubMedPubDate><PubMedPubDate 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