Volumetric diet through metabarcoding in a top predator

Metabarcoding has enhanced the ability to identify species contributing to wildlife diets, and blocking primers can improve prey detection by minimizing host DNA amplification. Relative read abundance (RRA) of prey species has been used as a semi-quantitative approach, based on the assumption that RRA reflects the actual dietary contribution of each species. However, this assumption has been questioned, and the impact of blocking primers on RRA estimates remains unclear. To assess the accuracy of RRA in estimating diet composition, we fed captive wolves six different diets and analyzed their scats using metabarcoding, both without and with four concentrations (5×, 10×, 15×, and 20×) of blocking primers. Our goal was to evaluate the validity of the semi-quantitative metabarcoding approach and the influence of blocking primers on the results.