# Metabarcoding provides accurate estimation of volumetric diet composition in a top predator despite interference from blocking primers

## TL;DR

This study shows that metabarcoding can accurately estimate the diet of wolves, even when using blocking primers, though some challenges remain.

## Contribution

The study demonstrates the accuracy of metabarcoding for diet estimation in wolves despite blocking primer interference.

## Key findings

- Relative read abundance accurately reflected dietary composition with an R2 of 0.815.
- Blocking primers increased false detections and reduced accuracy of diet estimates.
- Sample sizes over 30 scats improved diet estimation accuracy.

## Abstract

The study of diet is central to wildlife ecology, management and conservation. Metabarcoding enhanced the ability to identify species contributing to wildlife diets, and blocking primers can maximize prey detection. Relative read abundance (RRA) of different prey species has been used as semi-quantitative approach, assuming that RRA reflects species’ contributions to diet. We tested accuracy of RRA in diet estimation by feeding captive wolves six different diets. We analyzed samples both without and with four blocking primer concentrations (5, 10, 15 and 20×). RRA provided a highly accurate representation of the overall dietary composition, with best results obtained without blocking primer (0.775 ± 0.033; P < 0.001; R2 = 0.815). While the use of blocking the primer resulted in higher proportions of reads for diet items it did not increase the probability of detecting diet components. Moreover, the blocking primer led to increased detections of items not fed to wolves and produced slightly less accurate estimates of diet composition. Resampling indicated that sample sizes beyond 30 scats improved the accuracy of diet estimation. Our results are promising and support the use of metabarcoding to determine volumetric diet but caution and further research are necessary before extrapolating findings.

The online version contains supplementary material available at 10.1038/s41598-025-14837-9.