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

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; R = 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.