Assessing the influence of sample tagging and library preparation on DNA metabarcoding

Zizka, Vera M. A.; Elbrecht, Vasco; Macher, Jan‐Niklas; Leese, Florian

doi:10.1111/1755-0998.13018

Cited by 53 publications

(74 citation statements)

References 27 publications

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“…DNA metabarcoding with BF2/BR2 for biweekly data set: We applied a two-step PCR approach for the long-term biweekly data set (Zizka et al 2019). For the first PCR step, universal BF2/BR2 primers (Elbrecht and Leese 2017a) For bioinformatic analysis sequences were assigned to their original sample using JAMP v0.67 (https://github.com/VascoElbrecht/JAMP) (Elbrecht et al 2018).…”

Section: Methodsmentioning

confidence: 99%

Improved freshwater macroinvertebrate detection from eDNA through minimized non-target amplification

Leese

Sander

Buchner

et al. 2020

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Acknowledgments:We would like to thank Marlen Mährlein, Nathalie Kaffenberger, Cristina Hartmann-Fatu and Arne Beermann for help with sample collection or processing. Furthermore, we thank Jan-Niklas Macher for helpful discussions, Beatrice Kulawig for compiling the taxonomic data from the Rhine-Main-Observatory, and Martina Weiss for valuable comments on the structure of the manuscript. This work has been funded by a grant of the Bode Foundation to FL. This work has been conducted as part of COST (European Cooperation in Science and Technology) Action DNAqua-Net (CA15219). AbstractDNA metabarcoding of freshwater communities typically relies on PCR amplification of a fragment of the mitochondrial cytochrome c oxidase (COI) gene with degenerate primers. The advantage of COI is its taxonomic resolution and the availability of an extensive reference database.However, when universal primers are used on environmental DNA (eDNA) isolated from stream water, macroinvertebrate read and OTU numbers are typically "watered down", i.e. diluted, compared to whole specimen 'bulk samples' due to greater co-amplification of abundant nontarget taxa such as algae and bacteria. Because stream macroinvertebrate taxa are of prime importance for regulatory biomonitoring, more effective ways to capture their diversity via eDNA isolated from water are important. In this study, we aimed to improve macroinvertebrate assessment from eDNA by minimizing non-target amplification. Therefore, we generated data using universal primers BF2/BR2 throughout 15 months from a German Long-Term Ecological Research (LTER) site, the River Kinzig, to identify most abundant non-target taxa. Based on these data, we designed a new reverse primer (EPTDr2n) with 3'-specificity towards macrozoobenthic taxa and validated its specificity in silico together with universal forward primer fwhF2 using available data from GenBank and BOLD. We then performed in vitro tests using 20 eDNA samples taken in the Kinzig catchment. We found that the percentage of target reads was much higher for the new primer combination compared to two universal macrozoobenthic primer pairs, BF2/BR2 and fwhF2/fwhR2n (>99 % vs. 21.4 % and 41.25 %, respectively). Likewise, number of detected macroinvertebrate taxa was substantially higher (351 vs. 46 and 170, respectively) and exceeded the number of 257 taxa identified by expert taxonomists at nearby sites across two decades of sampling. While few taxa such as Turbellaria were not detected, we show that the optimized primer avoids the dilution problem and thus significantly improves macroinvertebrate detection for bioassessment and -monitoring.

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Section: Methodsmentioning

confidence: 99%

Improved freshwater macroinvertebrate detection from eDNA through minimized non-target amplification

Leese

Sander

Buchner

et al. 2020

Preprint

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“…In this study, we could only compare our metabarcoding results to the CPR data on copepods. We point out that comparisons of metabarcoding and CPR data should be interpreted with care, as the CPR samples only the top layers of the water column, and abundance inferred from metabarcoding data can be biased due to amplification and primers bias (Elbrecht & Leese, 2015Wangensteen et al, 2018;Zizka et al, 2019a). Further, comparing metabarcoding read abundance and number of specimens, as mostly provided by previous morphological studies, is challenging, as reads do not automatically correlate with specimen numbers.…”

Section: Discussionmentioning

confidence: 99%

“…15ng of DNA per sample was used for metabarcoding using the Leray-XT primers (313bp product length), which amplify a COI gene fragment of a wide range of marine metazoan taxa (Wangensteen et al, 2018). Samples were amplified using a two-step PCR protocol as commonly used for metabarcoding studies (Andruszkiewicz et al, 2017;Galan et al, 2018;Zizka et al, 2019a).…”

Section: Sampling Dna Extraction and Library Preparationmentioning

confidence: 99%

Metabarcoding reveals different zooplankton communities in northern and southern areas of the North Sea

Macher

Hoorn

Peijnenburg

et al. 2020

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Zooplankton are key players in marine ecosystems, linking primary production to higher trophic levels. The high abundance and high taxonomic diversity renders zooplankton ideal for biodiversity monitoring. However, taxonomic identification of the zooplankton assemblage is challenging due to its high diversity, subtle morphological differences and the presence of many meroplanktonic species, especially in coastal seas. Molecular techniques such as metabarcoding can help with rapid processing and identification of taxa in complex samples, and are therefore promising tools for identifying zooplankton communities. In this study, we applied metabarcoding of the mitochondrial cytochrome c oxidase I gene to zooplankton samples collected along a latitudinal transect in the North Sea, a shelf sea of the Atlantic Ocean. Northern regions of the North Sea are influenced by inflow of oceanic Atlantic waters, whereas the southern parts are characterised by more coastal waters. Our metabarcoding results indicated strong differences in zooplankton community composition between northern and southern areas of the North Sea, particularly in the classes Copepoda, Actinopterygii (ray-finned fishes) and Polychaeta. We compared these results to the known distributions of species reported in previous studies, and by comparing the abundance of copepods to data obtained from the Continuous Plankton Recorder (CPR). We found that our metabarcoding results are mostly congruent with the reported distribution and abundance patterns of zooplankton species in the North Sea. Our results highlight the power of metabarcoding to rapidly assess complex zooplankton samples, and we suggest that the technique could be used in future monitoring campaigns and biodiversity assessments.HighlightsZooplankton communities are different in northern and southern areas of the North SeaMetabarcoding results are consistent with known species distributions and abundanceMetabarcoding allows for fast identification of meroplanktonic species

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“…DNA was quantified with a Qubit Fluorometer (ds DNA BR Assay kit, Thermo Fisher Scientific, Beverly, USA) and adjusted to 25 ng/μl. A two-step PCR (for futher information see Zizka et al, 2019) was conducted with one technical (PCR) replicate per sample. The universal BF2/BR2 primers (Elbrecht and Leese, 2017) were used.…”

Section: Methodsmentioning

confidence: 99%

Can metabarcoding resolve intraspecific genetic diversity changes to environmental stressors? A test case using river macrozoobenthos

Zizka

Weiss

Leese

2020

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38Genetic diversity is the most basal level of biodiversity and determines the evolutionary capacity of 39 species to adapt to changing environments, yet it is typically neglected in routine biomonitoring and 40 stressor impact assessment. For a comprehensive analysis of stressor impacts on genetic diversity, it is 41 necessary to assess genetic variants simultaneously in many individuals and species. Such an assessment 42 is not as straight-forward and usually limited to one or few individual species. However, nowadays 43 species diversity can be assessed by analysing thousands of individuals of a community simultaneously 44 with DNA metabarcoding. Recent bioinformatic advances also allow for the extraction of exact 45 sequence variants (ESVs or haplotypes) in addition to Operational Taxonomic Units (OTUs). By using 46 this new capability, we here evaluated if the analysis of mitochondrial genetic diversity in addition to 47 species diversity can provide insights into responses of stream macrozoobenthic communities to 48 environmental stressors. For this purpose, we analysed macroinvertebrate bulk samples of three German 49 river systems with different stressor levels using DNA metabarcoding. While OTU and haplotype 50 number were negatively correlated with stressor impact, this association was not as clear when looking 51 at haplotype diversity. Here, stressor responses were only found for sensitive EPT (Ephemeroptera, 52Plecoptera, Trichoptera) taxa, and those exceedingly resistant to organic stress. An increase in haplotype 53 number per OTU and haplotype diversity of sensitive taxa was observed with an increase in ecosystem 54 quality and stability, while the opposite pattern was detected for pollution resistant taxa. However, this 55 pattern was less prominent than expected based on the strong differences in stressor intensity between 56 sites. To compare genetic diversity among river systems, only OTUs could be used, which were present 57 in all systems. As OTU composition differed strongly between the rivers, this led to the exclusion of a 58 high number of OTUs, especially in diverse river systems of good quality, which potentially diminished 59 the genetic diversity patterns. To better understand responses of genetic diversity to environmental 60 stressors for example in river ecosystems, it would be important to increase OTU overlap between sites 61 of comparisons, e.g. by sampling a narrower stressor gradient, and to perform calibrated studies 62 controlling for the number and individual genotypes. However, this pioneer study shows that the 63 extraction of haplotypes from DNA metabarcoding datasets is a promising tool to simultaneously assess 64 mitochondrial genetic diversity changes in response to environmental impacts for a metacommunity. 66 67Degradation, pollution, and exploitation of freshwater ecosystems has resulted in a drastic decline of 68 biodiversity (Vörösmarty et al., 2010; WWF, 2018). The magnitude of biodiversity loss depends on 69 stressor intensities as well as on resistance and re...

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Assessing the influence of sample tagging and library preparation on DNA metabarcoding

Cited by 53 publications

References 27 publications

Improved freshwater macroinvertebrate detection from eDNA through minimized non-target amplification

Improved freshwater macroinvertebrate detection from eDNA through minimized non-target amplification

Metabarcoding reveals different zooplankton communities in northern and southern areas of the North Sea

Can metabarcoding resolve intraspecific genetic diversity changes to environmental stressors? A test case using river macrozoobenthos

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