An assessment of minimum sequence copy thresholds for identifying and reducing the prevalence of artefacts in dietary metabarcoding data

Drake, Lorna E.; Cuff, Jordan P.; Young, Rebecca E.; Marchbank, Angela; Chadwick, Elizabeth Anna; Symondson, William O. C.

doi:10.1111/2041-210x.13780

Cited by 83 publications

(122 citation statements)

References 92 publications

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“…The Illumina Nano cartridge run generated 750,645 reads. Highthroughput sequencing data for the animal component of Telfair's skink diet followed the bioinformatic process of Drake et al (2021):…”

Section: Bioinformaticsmentioning

confidence: 99%

DNA metabarcoding reveals introduced species predominate in the diet of a threatened endemic omnivore, Telfair’s skink (Leiolopisma telfairii)

Tercel

Moorhouse‐Gann

Cuff

et al. 2021

Ecology and Evolution

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Introduced species can exert disproportionately negative effects on island ecosystems, but their potential role as food for native consumers is poorly studied. Telfair's skinks are endemic omnivores living on Round Island, Mauritius, a globally significant site of biodiversity conservation. We aimed to determine the dietary diversity and key trophic interactions of Telfair's skinks, whether introduced species are frequently consumed, and if diet composition changes seasonally between male and female skinks. We used DNA metabarcoding of skink fecal samples to identify animals (COI) and plants (ITS2) consumed by skinks. There were 389 dietary presence counts belonging to 77 dietary taxa found across the 73 Telfair's skink fecal samples. Introduced taxa were cumulatively consumed more frequently than other categories, accounting for 49.4% of all detections, compared to cryptogenic (20.6%), native (20.6%), and endemic taxa (9.5%). The most frequently consumed introduced species was the ant, Pheidole megacephala, present in 40% of samples. Blue latan palm, Latania loddigesii, was the most frequently consumed endemic species, present in 33% of samples but was only detected in the dry season, when fruits are produced. We found a strong seasonal difference in diet composition explained by the presence of certain plant species solely or primarily in one season and a marked increase in the consumption of animal prey in the dry season. Male and female skinks consumed several taxa at different frequencies. These results present a valuable perspective on the role of introduced species in the trophic network of their invaded ecosystem. Both native and introduced species provide nutritional resources for skinks, and this may have management implications in the context of species conservation and island restoration.

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Section: Bioinformaticsmentioning

confidence: 99%

DNA metabarcoding reveals introduced species predominate in the diet of a threatened endemic omnivore, Telfair’s skink (Leiolopisma telfairii)

Tercel

Moorhouse‐Gann

Cuff

et al. 2021

Ecology and Evolution

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“…All of these biases further compound issues surrounding the diversity of interactions detected and may result in large compartments of the ecological network being excluded, or connectance reduced between multiple layers (Figure 3). There are several key methods well‐suited to the elucidation of taxonomic biases in the metabarcoding process, from in silico simulations of primer performance and bias through to the use of mock communities (Braukmann et al., 2019; Drake et al., 2021; Elbrecht & Leese, 2016b). Understanding the biases present in the resultant data is crucial and can be used to rationalise some omissions or questionable results, but these biases are largely unavoidable.…”

Section: Metabarcoding Biasesmentioning

confidence: 99%

“…Stringent use of both negative and positive controls throughout the experimental process, and implementation of additional safeguards like spatial separation of pre‐ and post‐PCR samples and oil sealing of reactions (e.g. Kitson et al., 2019), are essential to ensure confidence in the study outcomes (Drake et al., 2021; Taberlet et al., 2018). A key aspect of this is the choice of bioinformatics process, which can profoundly affect the data output and thus any ecological inferences subsequently drawn (Clare et al., 2016).…”

Section: True Versus False Positivesmentioning

confidence: 99%

“…Following or even during bioinformatics, appropriate measures must be taken to remove false positive artefacts in the data output. These thresholds, termed minimum sequence copy thresholds, have traditionally involved the removal of read counts below an arbitrary value, but percentage sample read count thresholds and use of experimental controls to determine threshold values provide a more even solution which can more accurately remove false positives while maximising retention of target data (Drake et al., 2021). Instances in which these principles are not adhered to may introduce temporally, spatially or ecologically infeasible interactions and taxa into the networks.…”

Section: True Versus False Positivesmentioning

confidence: 99%

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Overcoming the pitfalls of merging dietary metabarcoding into ecological networks

et al. 2022

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The construction of increasingly detailed species interaction networks is extending the potential applications of network ecology, providing an opportunity to understand complex eco‐evolutionary interactions, ecosystem service provision and the impacts of environmental change on ecosystem functioning. Dietary metabarcoding is a rapidly growing tool increasingly used to construct ecological networks of trophic interactions, enabling the determination of individual animal diets including difficult‐to‐distinguish prey taxa and even for species where traditional dietary analyses are unsuitable (e.g. fluid feeders and small invertebrates). Several challenges, however, surround the use of dietary metabarcoding, especially when metabarcoding‐based interactions are merged with observation‐based species interaction data. We describe the difficulties surrounding the quantification of species interactions, sampling perspective discrepancy (i.e. zoocentric vs. phytocentric sampling), experimental biases, reference database omissions and assumptions regarding direct and indirect consumption events. These problems are not, however, insurmountable. Effective experimental design and data curation with appropriate attention paid to these problems renders the incorporation of dietary metabarcoding into ecological network analysis a powerful tool for the construction of highly resolved networks. Throughout, we discuss how these problems should be addressed when merging data to construct ecological networks.

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“…where S is the number of samples, T is the number of prey MOTUs, n i,k is the number of sequences of prey item i in sample k. We did not use an occurrence-based metric (i.e., presence/absence of taxa) because it often overestimates low-frequency taxa (including potential contamination) in sequencing data, and is sensitive to the data filter threshold (Deagle et al, 2019). To compare efficiency and consistency of the 12S and 16S primer sets, we used four filtering thresholds (0.1, 1, 3, and 5%) (Drake et al, 2021). Under each filter threshold, we obtained prey taxon richness based on RRA using the vegan package (Oksanen et al, 2020) in R v4.0.4 (R Core Team, 2021).…”

Section: Dietary Analysesmentioning

confidence: 99%

Assessing the Diet of a Predator Using a DNA Metabarcoding Approach

Wang

Zheng

et al. 2022

Front. Ecol. Evol.

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The diet of top predators is vital information needed to determine their ecological function and for their conservation management. However, the elusive habit and low population density of many predators constrains determination of their diets. While the morphological identification of scat contents is the traditional method, DNA metabarcoding has lately proven a more efficient and accurate method of identifying prey taxa. We applied DNA metabarcoding to analyzing the diet of the Eurasian otter (Lutra lutra), a top predator in freshwater ecosystems, using 12S and 16S rRNA mitochondrial primers target vertebrate prey. Diet did not vary among different data removal thresholds of 0.1, 1, 3, and 5%, comprising fishes (>90%), amphibians and birds (>2%), and occasionally mammals (<2%). Both 12S and 16S primers revealed similar otter diets, indicating that a single set of primers with a higher threshold is cost-effective for detecting the main prey taxa. Using 12S primers and a 5% threshold, we found no seasonal variation of otter diet in the Tangjiahe National Nature Reserve. A different prey community was found outside the reserve, which resulted in different prey composition for otters. However, prey taxon richness was not different between otters in- and outside the reserve. Otters preferred Schizothorax spp., the largest-sized fish species in the reserve, whereas they mainly preyed on Triplophysa bleekeri, a small-sized fish species, outside the reserve. Otters’ flexible feeding strategy reflect their high adaptability. However, greater human disturbance outside the reserve may present significant challenges to otters by altering prey communities and reducing prey profitability. Combining fecal DNA metabarcoding and local fish survey will provide opportunities for more detailed studies on the impact of different levels of human disturbances on prey communities and otters.

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An assessment of minimum sequence copy thresholds for identifying and reducing the prevalence of artefacts in dietary metabarcoding data

Cited by 83 publications

References 92 publications

DNA metabarcoding reveals introduced species predominate in the diet of a threatened endemic omnivore, Telfair’s skink (Leiolopisma telfairii)

DNA metabarcoding reveals introduced species predominate in the diet of a threatened endemic omnivore, Telfair’s skink (Leiolopisma telfairii)

Overcoming the pitfalls of merging dietary metabarcoding into ecological networks

Assessing the Diet of a Predator Using a DNA Metabarcoding Approach

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