Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Review
. 2023 Jan;23(1):41-51.
doi: 10.1111/1755-0998.13705. Epub 2022 Sep 10.

The predator problem and PCR primers in molecular dietary analysis: Swamped or silenced; depth or breadth?

Jordan P Cuff  1 , James J N Kitson  1 , David Hemprich-Bennett  2 , Maximillian P T G Tercel  3   4 , Samuel S Browett  5 , Darren M Evans  1
Affiliations
Review

The predator problem and PCR primers in molecular dietary analysis: Swamped or silenced; depth or breadth?

Jordan P Cuff et al. Mol Ecol Resour. 2023 Jan.

Abstract

Dietary metabarcoding has vastly improved our ability to analyse the diets of animals, but it is hampered by a plethora of technical limitations including potentially reduced data output due to the disproportionate amplification of the DNA of the focal predator, here termed "the predator problem". We review the various methods commonly used to overcome this problem, from deeper sequencing to exclusion of predator DNA during PCR, and how they may interfere with increasingly common multipredator-taxon studies. We suggest that multiprimer approaches with an emphasis on achieving both depth and breadth of prey detections may overcome the issue to some extent, although multitaxon studies require further consideration, as highlighted by an empirical example. We also review several alternative methods for reducing the prevalence of predator DNA that are conceptually promising but require additional empirical examination. The predator problem is a key constraint on molecular dietary analyses but, through this synthesis, we hope to guide researchers in overcoming this in an effective and pragmatic way.

Keywords: DNA metabarcoding; amplification; food webs; molecular analysis; trophic interactions.

PubMed Disclaimer

Conflict of interest statement

There are no conflicts of interest to declare.

Figures

FIGURE 1
FIGURE 1
The hypothetical degradation of DNA throughout the digestive process preluding whole body DNA extraction (left), stomach flush or regurgitate DNA extraction (centre), and faecal DNA extraction (right). Line graphs below illustrate DNA prevalence over time. In whole bodies, prey DNA degrades linearly, but predator DNA remains constantly prevalent. In regurgitates, DNA prevalence will decrease following removal from the predator's body and will then degrade alongside the prey DNA. In faeces, predator DNA will be less prevalent following defecation, and will then degrade with prey DNA, which will have degraded throughout the entire digestive process. Figure created in Biorender.com
FIGURE 2
FIGURE 2
General PCR primers will amplify a broad taxonomic range, but most of the PCR product will be the predator itself, resulting in some species being omitted in the sequencing output. Exclusion primers will avoid amplification of the predator, but also some prey groups, particularly those phylogenetically close to the predator (e.g., the flour beetle being eaten by the carabid beetle in this image). Figure created in Biorender.com
FIGURE 3
FIGURE 3
The percentage of reads attributed to predator and prey for each spider genus across two primer pairs used by Cuff, Tercel, et al. (2022). Colours distinguish between predator (red) and prey (blue) reads, and read counts are given as percentages of the total read count per sample. Figure originally presented by Cuff, Tercel, et al. (2022)
FIGURE 4
FIGURE 4
Nonmetric multidimensional scaling showing higher similarity of dietary data from the same primer pairs (colours; red and blue denoting general and exclusion primers, respectively) than within samples (points linked by black lines belong to the same sample). Creation of this figure is described in Appendix S1
See this image and copyright information in PMC

References

    1. Alberdi, A. , Aizpurua, O. , Bohmann, K. , Lynggaard, C. , Nielsen, M. , Thomas, M. , & Gilbert, P. (2018). Promises and pitfalls of using high ‐ throughput sequencing for diet analysis. Molecular Ecology Resources, 19(2), 327–348. 10.1111/1755-0998.12960 - DOI - PubMed
    1. Ammann, L. , Moorhouse‐Gann, R. , Cuff, J. , Bertrand, C. , Mestre, L. , Hidalgo, N. P. , Ellison, A. , Herzog, F. , Entling, M. H. , Albrecht, M. , & Symondson, W. O. C. (2020). Insights into aphid prey consumption by ladybirds: Optimising field sampling methods and primer design for high throughput sequencing. PLoS One, 15(7), e0235054. 10.1371/journal.pone.0235054 - DOI - PMC - PubMed
    1. Aylward, M. L. , Sullivan, A. P. , Perry, G. H. , Johnson, S. E. , & Louis, E. E. J. (2018). An environmental DNA sampling method for aye‐ayes from their feeding traces. Ecology and Evolution, 8(18), 9229–9240. - PMC - PubMed
    1. Baloğlu, B. , Chen, Z. , Elbrecht, V. , Braukmann, T. , MacDonald, S. , & Steinke, D. (2021). A workflow for accurate metabarcoding using nanopore MinION sequencing. Methods in Ecology and Evolution, 12(5), 794–804.
    1. Batuecas, I. , Alomar, O. , Castañe, C. , Piñol, J. , Boyer, S. , Gallardo‐Montoya, L. , & Agustí, N. (2022). Development of a multi‐primer metabarcoding approach to understanding trophic interactions in agroecosystems. Insect Science, 29(4), 1195–1210. - PubMed

LinkOut - more resources