# Dietary DNA Metabarcoding From Animal Fecal Samples

## TL;DR

This paper provides a detailed guide for using DNA from animal feces to study their diets, covering fieldwork, lab techniques, and data analysis.

## Contribution

The paper introduces a practical, step-by-step workflow for dietary DNA metabarcoding using QIIME2 and Illumina sequencing.

## Key findings

- A complete protocol for preparing sequencing libraries from fecal DNA is described.
- A QIIME2-based bioinformatics pipeline is outlined for processing dietary DNA sequences.
- Key methodological considerations and statistical approaches for diet data analysis are highlighted.

## Abstract

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