788
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Increased efficiency in identifying mixed pollen samples by meta-barcoding with a dual-indexing approach

      research-article

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Background

          Meta-barcoding of mixed pollen samples constitutes a suitable alternative to conventional pollen identification via light microscopy. Current approaches however have limitations in practicability due to low sample throughput and/or inefficient processing methods, e.g. separate steps for amplification and sample indexing.

          Results

          We thus developed a new primer-adapter design for high throughput sequencing with the Illumina technology that remedies these issues. It uses a dual-indexing strategy, where sample-specific combinations of forward and reverse identifiers attached to the barcode marker allow high sample throughput with a single sequencing run. It does not require further adapter ligation steps after amplification. We applied this protocol to 384 pollen samples collected by solitary bees and sequenced all samples together on a single Illumina MiSeq v2 flow cell. According to rarefaction curves, 2,000–3,000 high quality reads per sample were sufficient to assess the complete diversity of 95% of the samples. We were able to detect 650 different plant taxa in total, of which 95% were classified at the species level. Together with the laboratory protocol, we also present an update of the reference database used by the classifier software, which increases the total number of covered global plant species included in the database from 37,403 to 72,325 (93% increase).

          Conclusions

          This study thus offers improvements for the laboratory and bioinformatical workflow to existing approaches regarding data quantity and quality as well as processing effort and cost-effectiveness. Although only tested for pollen samples, it is furthermore applicable to other research questions requiring plant identification in mixed and challenging samples.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s12898-015-0051-y) contains supplementary material, which is available to authorized users.

          Related collections

          Most cited references25

          • Record: found
          • Abstract: not found
          • Book Chapter: not found

          AMPLIFICATION AND DIRECT SEQUENCING OF FUNGAL RIBOSOMAL RNA GENES FOR PHYLOGENETICS

            Bookmark
            • Record: found
            • Abstract: found
            • Article: found
            Is Open Access

            Validation of the ITS2 Region as a Novel DNA Barcode for Identifying Medicinal Plant Species

            Background The plant working group of the Consortium for the Barcode of Life recommended the two-locus combination of rbcL + matK as the plant barcode, yet the combination was shown to successfully discriminate among 907 samples from 550 species at the species level with a probability of 72%. The group admits that the two-locus barcode is far from perfect due to the low identification rate, and the search is not over. Methodology/Principal Findings Here, we compared seven candidate DNA barcodes (psbA-trnH, matK, rbcL, rpoC1, ycf5, ITS2, and ITS) from medicinal plant species. Our ranking criteria included PCR amplification efficiency, differential intra- and inter-specific divergences, and the DNA barcoding gap. Our data suggest that the second internal transcribed spacer (ITS2) of nuclear ribosomal DNA represents the most suitable region for DNA barcoding applications. Furthermore, we tested the discrimination ability of ITS2 in more than 6600 plant samples belonging to 4800 species from 753 distinct genera and found that the rate of successful identification with the ITS2 was 92.7% at the species level. Conclusions The ITS2 region can be potentially used as a standard DNA barcode to identify medicinal plants and their closely related species. We also propose that ITS2 can serve as a novel universal barcode for the identification of a broader range of plant taxa.
              Bookmark
              • Record: found
              • Abstract: not found
              • Article: not found

              Foraging ranges of solitary bees

                Bookmark

                Author and article information

                Contributors
                wiebke.sickel@uni-wuerzburg.de
                markus.ankenbrand@uni-wuerzburg.de
                grimmer@biozentrum.uni-wuerzburg.de
                andrea.holzschuh@uni-wuerzburg.de
                stephan.haertel@uni-wuerzburg.de
                jonathan.lanzen@uni-wuerzburg.de
                ingolf.steffan@uni-wuerzburg.de
                a.keller@biozentrum.uni-wuerzburg.de
                Journal
                BMC Ecol
                BMC Ecol
                BMC Ecology
                BioMed Central (London )
                1472-6785
                22 July 2015
                22 July 2015
                2015
                : 15
                : 20
                Affiliations
                Department of Animal Ecology and Tropical Biology, Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany
                Author information
                http://orcid.org/0000-0001-5716-3634
                Article
                51
                10.1186/s12898-015-0051-y
                4509727
                26194794
                724d0b36-9350-4ad0-bbe0-9a8787af82f5
                © Sickel et al. 2015

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 15 April 2015
                : 25 June 2015
                Categories
                Methodology Article
                Custom metadata
                © The Author(s) 2015

                Ecology
                dna barcoding,high throughput sequencing,illumina miseq platform,its2,next generation sequencing,ngs,osmia,palynology,pollination ecology

                Comments

                Comment on this article