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Comparison and validation of some ITS primer pairs useful for fungal metabarcoding studies

Op De Beeck, Michiel LU ; Lievens, Bart; Busschaert, Pieter; Declerck, Stéphan; Vangronsveld, Jaco and Colpaert, Jan V (2014) In PLoS ONE 9(6).
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... (More)

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 in silico 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 in silico 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.

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published
subject
keywords
Belgium, Computer Simulation, DNA Barcoding, Taxonomic/methods, DNA Primers/genetics, DNA, Fungal/genetics, DNA, Ribosomal Spacer/genetics, Fungi/classification, Polymerase Chain Reaction, Reproducibility of Results, Soil Microbiology
in
PLoS ONE
volume
9
issue
6
pages
11 pages
publisher
Public Library of Science
external identifiers
  • scopus:84903128093
ISSN
1932-6203
DOI
10.1371/journal.pone.0097629
language
English
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no
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7af65d3a-2a29-4dc6-9c15-439dca21880f
date added to LUP
2019-03-05 15:53:01
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2019-06-16 05:13:15
@article{7af65d3a-2a29-4dc6-9c15-439dca21880f,
  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 in silico 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 in silico 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>},
  articleno    = {e97629},
  author       = {Op De Beeck, Michiel and Lievens, Bart and Busschaert, Pieter and Declerck, Stéphan and Vangronsveld, Jaco and Colpaert, Jan V},
  issn         = {1932-6203},
  keyword      = {Belgium,Computer Simulation,DNA Barcoding, Taxonomic/methods,DNA Primers/genetics,DNA, Fungal/genetics,DNA, Ribosomal Spacer/genetics,Fungi/classification,Polymerase Chain Reaction,Reproducibility of Results,Soil Microbiology},
  language     = {eng},
  month        = {06},
  number       = {6},
  pages        = {11},
  publisher    = {Public Library of Science},
  series       = {PLoS ONE},
  title        = {Comparison and validation of some ITS primer pairs useful for fungal metabarcoding studies},
  url          = {http://dx.doi.org/10.1371/journal.pone.0097629},
  volume       = {9},
  year         = {2014},
}