An experimental design for obtaining DNA of a target species and its diet from a single non-invasive genetic protocol

Next-generation sequencing technology has enabled accurate insights into the diet of wildlife species. The protocols for faecal sample collection and DNA extraction for diet analysis have differed from those focusing on target species, even in most studies combining questions on both aspects. We designed an experiment to evaluate two protocols using 11 parameters and select a single one that will generate both target species (Asiatic wild ass, Equus hemionus, in Israel) and diet DNA, as an effective strategy to minimise time, effort, and cost without hampering efficiency. In Protocol A, we swabbed the outer surface of faecal boluses and extracted DNA using a Stool Kit, while for Protocol B, we homogenised faecal matter from inside the bolus followed by extraction using a Powersoil Kit. Protocol A performed significantly better for four parameters, which included, for the target species, microsatellite amplification success and the quantity of the GAPDH gene; and for its diet, the number of exact sequence variants (ESVs) obtained at genus level and plant genus richness. However, there was no significant difference in the amplification success of sex-linked and plant markers, total reads at genus level, number of genera obtained and plant genus composition. Although we chose Protocol A, both protocols yielded results for the target species and its diet, demonstrating that one single protocol can be used for both purposes, although a pilot study is recommended to optimise the protocol for specific systems. This strategy may also be useful for studies combining target species and their gut microbiome and parasitic load.