The precautionary principle and dietary DNA metabarcoding: Commonly used abundance thresholds change ecological interpretation

Littleford‐Colquhoun, Bethan L.; Freeman, Patrick T.; Sackett, Violet; Tulloss, Camille V.; McGarvey, Lauren M.; Geremia, Chris; Kartzinel, Tyler R.

doi:10.1111/mec.16352

Cited by 26 publications

(60 citation statements)

References 68 publications

(105 reference statements)

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“…In this study, we applied a stringent filtering of the data to avoid spurious DNA, using percentual and absolute thresholds. It has been argued that arbitrary minimum copy thresholds might omit true sequences (Littleford‐Colquhoun et al, 2022 ) and that percentual thresholds were more suitable in case of uneven sequencing depths (Drake et al, 2022 ). To avoid the generation of supplementary biases, it is recommended to normalize PCR amplicons before pooling.…”

Section: Discussionmentioning

confidence: 99%

Focal vs. fecal: Seasonal variation in the diet of wild vervet monkeys from observational and DNA metabarcoding data

Brun

Schneider

Mas‐Carrió

et al. 2022

Ecology and Evolution

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Assessing the diet of wild animals reveals valuable information about their ecology and trophic relationships that may help elucidate dynamic interactions in ecosystems and forecast responses to environmental changes. Advances in molecular biology provide valuable research tools in this field. However, comparative empirical research is still required to highlight strengths and potential biases of different approaches. Therefore, this study compares environmental DNA and observational methods for the same study population and sampling duration. We employed DNA metabarcoding assays targeting plant and arthropod diet items in 823 fecal samples collected over 12 months in a wild population of an omnivorous primate, the vervet monkey ( Chlorocebus pygerythrus ). DNA metabarcoding data were subsequently compared to direct observations. We observed the same seasonal patterns of plant consumption with both methods; however, DNA metabarcoding showed considerably greater taxonomic coverage and resolution compared to observations, mostly due to the construction of a local plant DNA database. We found a strong effect of season on variation in plant consumption largely shaped by the dry and wet seasons. The seasonal effect on arthropod consumption was weaker, but feeding on arthropods was more frequent in spring and summer, showing overall that vervets adapt their diet according to available resources. The DNA metabarcoding assay outperformed also direct observations of arthropod consumption in both taxonomic coverage and resolution. Combining traditional techniques and DNA metabarcoding data can therefore not only provide enhanced assessments of complex diets and trophic interactions to the benefit of wildlife conservationists and managers but also opens new perspectives for behavioral ecologists studying whether diet variation in social species is induced by environmental differences or might reflect selective foraging behaviors.

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Section: Discussionmentioning

confidence: 99%

Focal vs. fecal: Seasonal variation in the diet of wild vervet monkeys from observational and DNA metabarcoding data

Brun

Schneider

Mas‐Carrió

et al. 2022

Ecology and Evolution

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“…When low sample sizes are used, outcomes should be considered with additional scrutiny, particularly considering how stochastic outliers and false positives may overlap with greater frequency and how this may skew ecological outcomes. In their article, Littleford-Colquhoun et al (2022) argue that dietary metabarcoding data are very sensitive to changes in MSCT levels, but their analyses may also show that dietary data arising from a small sample size are very sensitive to changes in MSCT levels. Littleford-Colquhoun et al (2022) have stimulated a longoverdue discussion about data clean-up, its imperfections and the lack of clear consensus in the community, particularly as we transition to increasingly multiconsumer dietary studies.…”

Section: Other Advan Ce S From the Discussion Of The Prec Auti Onary ...mentioning

confidence: 99%

“…In their article, Littleford-Colquhoun et al (2022) argue that dietary metabarcoding data are very sensitive to changes in MSCT levels, but their analyses may also show that dietary data arising from a small sample size are very sensitive to changes in MSCT levels. Littleford-Colquhoun et al (2022) have stimulated a longoverdue discussion about data clean-up, its imperfections and the lack of clear consensus in the community, particularly as we transition to increasingly multiconsumer dietary studies. This discussion is critical for ensuring the long-term robustness, accessibility and accuracy of metabarcoding, but additional perspectives are necessary and we disagree that the only valid use of MSCTs are studies simply building a species list.…”

Section: Other Advan Ce S From the Discussion Of The Prec Auti Onary ...mentioning

confidence: 99%

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The complex epistemological challenge of data curation in dietary metabarcoding: Comment on “The precautionary principle and dietary DNA metabarcoding: Commonly used abundance thresholds change ecological interpretation” by Littleford‐Colquhoun et al. (2022)

Tercel

Cuff

2022

Molecular Ecology

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In their article, Littleford‐Colquhoun et al. (2022) advise against using arbitrary relative read abundance (RRA) thresholds (i.e., minimum sequence copy thresholds) for removing low‐abundance sequences since they can increase false negative rates in dietary DNA metabarcoding data sets. The main criticisms presented against these widespread methods are that they (i) are arbitrary, often existing as standard values or defined based on researcher‐selected delineations, (ii) are subjective, varying between studies and contexts, and, most problematically, (iii) result in the exclusion of true positives, particularly rarely consumed taxa, to the detriment of ecological insight. We commend the authors for presenting a refreshing and timely perspective on this often neglected topic, which is certainly in need of greater discussion following over a decade of significant advances in dietary metabarcoding. In this complex epistemological problem of false positives versus false negatives, we feel that several of the points raised deserve additional discussion. We address these aspects below, including measured approaches to data filtration and consistent representation of RRAs, and we welcome any further discourse to solidify or refute the concepts therein.

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“…Recent work has found that the practice of removing rare sequences distorts the accurate compositional representation in a sample, making it more difficult to detect differences between treatments and disproportionately impacting samples with lower sequence depth (38). Further, this practice may be problematic for some research goals, particularly among studies investigating the rare biosphere, dietary profiles, or topology of trophic networks (39, 40). In these investigations it is preferred to identify those sequences that are not ubiquitous among samples and are distant to BLAST matches.…”

Section: Discussionmentioning

confidence: 99%

Multi-factorial examination of amplicon sequencing workflows from sample preparation to bioinformatic analysis

Wolfe

Wright

2022

Preprint

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The development of sequencing technologies to evaluate bacterial microbiota composition has allowed new insights into the importance of microbial ecology. However, the variety of methodologies used among amplicon sequencing workflows leads to uncertainty about best practices as well as reproducibility and replicability among microbiome studies. Using a bacterial mock community composed of 37 soil isolates, we performed a comprehensive methodological evaluation of 540 workflows, each with a different combination of methodological factors spanning sample preparation to bioinformatic analysis to define sources of artifacts that affect sensitivity, specificity, and biases in the resulting compositional profiles. Of the 540 workflows examined, those using the V4-V4 primer set enabled the highest level of concordance between the original mock community and resulting microbiome sequence composition. Use of a high-fidelity polymerase, or a lower-fidelity polymerase with increased PCR elongation time limited chimera formation. Bioinformatic pipelines presented a trade-off between the fraction of distinct community members identified (sensitivity) and fraction of correct sequences (specificity). DADA2 and QIIME2 assembled V4-V4 reads amplified by Taq polymerase resulted in the highest specificity (100%), but only identified 52% of mock community members. Using mothur to assemble and denoise V4-V4 reads resulted in detection of 75% of mock community members among the resulting sequences, albeit with marginally lower specificity (99.5%). Optimization of microbiome workflows is critical for accuracy and to support reproducibility and replicability among microbiome studies. These aspects will help reveal the guiding principles of microbial ecology and impact the translation of microbiome research to human and environmental health.

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The precautionary principle and dietary DNA metabarcoding: Commonly used abundance thresholds change ecological interpretation

Abstract: This is an open access article under the terms of the Creat ive Commo ns Attri bution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Cited by 26 publications

References 68 publications

Focal vs. fecal: Seasonal variation in the diet of wild vervet monkeys from observational and DNA metabarcoding data

Focal vs. fecal: Seasonal variation in the diet of wild vervet monkeys from observational and DNA metabarcoding data

The complex epistemological challenge of data curation in dietary metabarcoding: Comment on “The precautionary principle and dietary DNA metabarcoding: Commonly used abundance thresholds change ecological interpretation” by Littleford‐Colquhoun et al. (2022)

Multi-factorial examination of amplicon sequencing workflows from sample preparation to bioinformatic analysis

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