Dietary DNA Metabarcoding From Animal Fecal Samples.

Fecal DNA metabarcoding is a powerful tool for examining animal diets with unprecedented resolution, offering insights into ecological patterns shaped by trophic interactions. As a result, dietary metabarcoding has become widely applied across ecology, evolution, behavior, and conservation. This article provides a practical guide to the key steps involved in metabarcoding animal fecal samples, from field collection and storage through to laboratory processes, such as DNA extraction, PCR amplification, and sequencing library preparation. It also outlines a bioinformatics workflow using the open-source QIIME2 platform to filter, error-correct, and assign taxonomy to dietary DNA sequences. We present key considerations for study design, highlighting potential caveats and limitations to enable researchers to make informed methodological choices. In addition, we offer guidance on the statistical analysis of diet data, including generalized linear models, multivariate analyses, and network analyses. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Metabarcoding library preparation of animal fecal dietary DNA for Illumina MiSeq sequencing Support Protocol 1: Making a SpeedBeads solution Support Protocol 2: Calibrating the SpeedBeads solution Basic Protocol 2: QIIME2 bioinformatics workflow for metabarcoded dietary DNA.