Evaluating the Quantitative Accuracy and Application of DNA Metabarcoding for Dietary Reconstruction in Ruminants

Area of Science:

• Ecology
• Molecular Biology
• Animal Science

Background:

• DNA metabarcoding is a powerful tool for non-invasive dietary analysis.
• Quantitative accuracy and biases are known limitations, particularly for herbivores due to plant material.
• Understanding these limitations is crucial for accurate ecological and management applications.

Purpose of the Study:

• To evaluate DNA metabarcoding accuracy for sheep diets using ITS2 and trnL markers.
• To assess the detectability and proportional representation of Medicago sativa in sheep diets.
• To identify biases associated with forage digestibility and marker choice.

Main Methods:

• Controlled feeding trials with sheep using high and low digestibility forages.
• DNA metabarcoding analysis of fecal samples using ITS2 and trnL plant DNA barcodes.
• Quantification of Medicago sativa inclusion levels (1%, 5%, 10%) and sequence read representation.

Main Results:

• ITS2 offered higher species resolution; trnL provided broader taxonomic coverage.
• Both markers differentiated diet treatments, but fecal DNA proportions deviated from vegetation input, especially with low-digestibility diets.
• Medicago sativa was detected at 1% but consistently overrepresented in sequence data.

Conclusions:

• DNA metabarcoding shows strengths and limitations for herbivore diet studies.
• Marker selection and differential digestion significantly influence quantitative accuracy.
• Multi-marker approaches and calibration are essential for reliable quantitative dietary studies in herbivores.