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      Scaling up DNA metabarcoding for freshwater macrozoobenthos monitoring

      1 , 1 , 2
      Freshwater Biology
      Wiley

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          Scrutinizing key steps for reliable metabarcoding of environmental samples

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            A universal DNA mini-barcode for biodiversity analysis

            Background The goal of DNA barcoding is to develop a species-specific sequence library for all eukaryotes. A 650 bp fragment of the cytochrome c oxidase 1 (CO1) gene has been used successfully for species-level identification in several animal groups. It may be difficult in practice, however, to retrieve a 650 bp fragment from archival specimens, (because of DNA degradation) or from environmental samples (where universal primers are needed). Results We used a bioinformatics analysis using all CO1 barcode sequences from GenBank and calculated the probability of having species-specific barcodes for varied size fragments. This analysis established the potential of much smaller fragments, mini-barcodes, for identifying unknown specimens. We then developed a universal primer set for the amplification of mini-barcodes. We further successfully tested the utility of this primer set on a comprehensive set of taxa from all major eukaryotic groups as well as archival specimens. Conclusion In this study we address the important issue of minimum amount of sequence information required for identifying species in DNA barcoding. We establish a novel approach based on a much shorter barcode sequence and demonstrate its effectiveness in archival specimens. This approach will significantly broaden the application of DNA barcoding in biodiversity studies.
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              Tag jumps illuminated--reducing sequence-to-sample misidentifications in metabarcoding studies.

              Metabarcoding of environmental samples on second-generation sequencing platforms has rapidly become a valuable tool for ecological studies. A fundamental assumption of this approach is the reliance on being able to track tagged amplicons back to the samples from which they originated. In this study, we address the problem of sequences in metabarcoding sequencing outputs with false combinations of used tags (tag jumps). Unless these sequences can be identified and excluded from downstream analyses, tag jumps creating sequences with false, but already used tag combinations, can cause incorrect assignment of sequences to samples and artificially inflate diversity. In this study, we document and investigate tag jumping in metabarcoding studies on Illumina sequencing platforms by amplifying mixed-template extracts obtained from bat droppings and leech gut contents with tagged generic arthropod and mammal primers, respectively. We found that an average of 2.6% and 2.1% of sequences had tag combinations, which could be explained by tag jumping in the leech and bat diet study, respectively. We suggest that tag jumping can happen during blunt-ending of pools of tagged amplicons during library build and as a consequence of chimera formation during bulk amplification of tagged amplicons during library index PCR. We argue that tag jumping and contamination between libraries represents a considerable challenge for Illumina-based metabarcoding studies, and suggest measures to avoid false assignment of tag jumping-derived sequences to samples.
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                Author and article information

                Journal
                Freshwater Biology
                Freshw Biol
                Wiley
                0046-5070
                1365-2427
                December 10 2018
                December 10 2018
                Affiliations
                [1 ]Centre for Biodiversity GenomicsUniversity of Guelph Guelph Ontario Canada
                [2 ]Department of Integrative BiologyUniversity of Guelph Guelph Ontario Canada
                Article
                10.1111/fwb.13220
                7b5e8314-e1cd-4899-b250-f1b6a04adb6c
                © 2018

                http://doi.wiley.com/10.1002/tdm_license_1.1

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