An experimental design for obtaining DNA of a focal 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 extraction of DNA for diet analysis have been different from those focusing on focal species DNA, even in studies combining questions on a focal species and its diet. We designed an experiment to evaluate two protocols and select a single one that will generate both focal species (Asiatic wild ass, Equus hemionus, in Israel) and diet DNA, as an effective strategy to minimize time, efforts, 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 most of the measures, which included, for the focal species, microsatellite amplification success and the quantity of the GAPDH gene; and for its diet, amplification success of a chloroplast barcoding marker, trnL, number of reads, and plant genus richness and diversity. However, there was no significant difference for the amplification success of a sex-linked marker and plant genus composition. Both protocols yielded results for the focal 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 focal species and their gut microbiome and parasitic load.