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      Comparison and Validation of Some ITS Primer Pairs Useful for Fungal Metabarcoding Studies

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          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 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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          Most cited references24

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          454 Pyrosequencing analyses of forest soils reveal an unexpectedly high fungal diversity.

          * Soil fungi play a major role in ecological and biogeochemical processes in forests. Little is known, however, about the structure and richness of different fungal communities and the distribution of functional ecological groups (pathogens, saprobes and symbionts). * Here, we assessed the fungal diversity in six different forest soils using tag-encoded 454 pyrosequencing of the nuclear ribosomal internal transcribed spacer-1 (ITS-1). No less than 166 350 ITS reads were obtained from all samples. In each forest soil sample (4 g), approximately 30 000 reads were recovered, corresponding to around 1000 molecular operational taxonomic units. * Most operational taxonomic units (81%) belonged to the Dikarya subkingdom (Ascomycota and Basidiomycota). Richness, abundance and taxonomic analyses identified the Agaricomycetes as the dominant fungal class. The ITS-1 sequences (73%) analysed corresponded to only 26 taxa. The most abundant operational taxonomic units showed the highest sequence similarity to Ceratobasidium sp., Cryptococcus podzolicus, Lactarius sp. and Scleroderma sp. * This study validates the effectiveness of high-throughput 454 sequencing technology for the survey of soil fungal diversity. The large proportion of unidentified sequences, however, calls for curated sequence databases. The use of pyrosequencing on soil samples will accelerate the study of the spatiotemporal dynamics of fungal communities in forest ecosystems.
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            Bias in template-to-product ratios in multitemplate PCR.

            Bias introduced by the simultaneous amplification of specific genes from complex mixtures of templates remains poorly understood. To explore potential causes and the extent of bias in PCR amplification of 16S ribosomal DNAs (rDNAs), genomic DNAs of two closely and one distantly related bacterial species were mixed and amplified with universal, degenerate primers. Quantification and comparison of template and product ratios showed that there was considerable and reproducible overamplification of specific templates. Variability between replicates also contributed to the observed bias but in a comparatively minor way. Based on these initial observations, template dosage and differences in binding energies of permutations of the degenerate, universal primers were tested as two likely causes of this template-specific bias by using 16S rDNA templates modified by site-directed mutagenesis. When mixtures of mutagenized templates containing AT- and GC-rich priming sites were used, templates containing the GC-rich permutation amplified with higher efficiency, indicating that different primer binding energies may to a large extent be responsible for overamplification. In contrast, gene copy number was found to be an unlikely cause of the observed bias. Similarly, amplification from DNA extracted from a natural community to which different amounts of genomic DNA of a single bacterial species were added did not affect relative product ratios. Bias was reduced considerably by using high template concentrations, by performing fewer cycles, and by mixing replicate reaction preparations.
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              Quantifying microbial communities with 454 pyrosequencing: does read abundance count?

              Pyrosequencing technologies have revolutionized how we describe and compare complex microbial communities. In 454 pyrosequencing data sets, the abundance of reads pertaining to taxa or phylotypes is commonly interpreted as a measure of genic or taxon abundance, useful for quantitative comparisons of community similarity. Potentially systematic biases inherent in sample processing, amplification and sequencing, however, may alter read abundance and reduce the utility of quantitative metrics. Here, we examine the relationship between read abundance and biological abundance in a sample of house dust spiked with known quantities and identities of fungi along a dilution gradient. Our results show one order of magnitude differences in read abundance among species. Precision of quantification within species along the dilution gradient varied from R(2) of 0.96-0.54. Read-quality based processing stringency profoundly affected the abundance of one species containing long homopolymers in a read orientation-biased manner. Order-level composition of background environmental fungal communities determined from pyrosequencing data was comparable with that derived from cloning and Sanger sequencing and was not biased by read orientation. We conclude that read abundance is approximately quantitative within species, but between-species comparisons can be biased by innate sequence structure. Our results showed a trade off between sequence quality stringency and quantification. Careful consideration of sequence processing methods and community analyses are warranted when testing hypotheses using read abundance data. © 2010 Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                16 June 2014
                : 9
                : 6
                : e97629
                Affiliations
                [1 ]Centre for Environmental Sciences, Hasselt University, Hasselt, Limburg, Belgium
                [2 ]Department of Microbial and Molecular Systems (M2S), Catholic University of Leuven, Antwerp, Belgium
                [3 ]Earth & Life Institute, Catholic University of Louvain, Louvain-la-Neuve, Walloon Brabant, Belgium
                University of New South Wales, Australia
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                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.

                Article
                PONE-D-14-00713
                10.1371/journal.pone.0097629
                4059633
                24933453
                18e3644c-84ff-4b5d-8cd9-49ff1bf3c7bd
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 17 January 2014
                : 21 April 2014
                Page count
                Pages: 11
                Funding
                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.
                Categories
                Research Article
                Biology and Life Sciences
                Ecology
                Plant Ecology
                Plant-Environment Interactions
                Biodiversity
                Community Ecology
                Microbial Ecology
                Microbiology
                Plant Microbiology
                Mycology
                Organisms
                Fungi
                Ecology and Environmental Sciences
                Soil Science
                Soil Ecology

                Uncategorized
                Uncategorized

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