Metabarcoding the Diet of Terrestrial Carnivore Species

Metabarcoding of fecal samples is a powerful, non-invasive approach for investigating the feeding ecology of carnivores, revealing prey diversity and unexpected dietary components with greater resolution than traditional methods. However, the approach remains technically demanding, as challenges and potential biases arise at every stage, from scat collection and DNA extraction to primer selection, sequencing, and data interpretation. Methodological details for these steps are often scattered across studies, limiting reproducibility and accessibility for ecologists. Here, we present a comprehensive field-to-sequencer workflow for dietary metabarcoding of terrestrial carnivores using Oxford Nanopore Technologies (ONT), covering all stages from sample collection to ecological interpretation. Drawing on field-collected scats of brown (Parahyaena brunnea) and spotted hyenas (Crocuta crocuta) across arid and semi-arid savannas in Botswana, we illustrate practical decisions, technical considerations, and common pitfalls encountered throughout the process. By integrating field, laboratory, and bioinformatic components into a single, accessible framework, this paper provides a pragmatic reference for ecologists aiming to design robust, transparent, and comparable studies of carnivore diet composition.