DNA extraction and species identification
DNA from collected scats were initially extracted using the QIAamp Fast DNA Stool Mini Kits (Qiagen, Hilden, Germany) following the manufacturer protocol. Subsequent re-extractions were performed using a modified protocol to increase DNA quantity and included the following additional steps: 1) duplicate samples were taken from each scat and combined downstream, 2) samples were incubated overnight at 60°C prior to homogenization, 3) 100ul of Buffer ATE was used for eluting DNA, 4) samples were incubated at 60°C for 5 minutes prior to centrifuging for elution, and 5) eluate was pipetted back onto the spin column, incubated for 5 minutes, and centrifuged again. To prevent cross-contamination, scats were extracted in a dedicated low-quality DNA processing lab and gloves were changed between each sample. We designed species-specific primers and probes for bobcat (mitochondrial genome subunit 5 gene; GenBank: KP202285.1) and coyote (Canis latrans) (mitochondrial genome isolate 1 USA; GenBank: DQ480510.1) using Primer3 (Koressaar and Remm 2007; Untergasser et al. 2012; Koressaar et al. 2018) and checked for species specificity using NCBI’s Primer-BLAST (Ye et al. 2012) (Table 1). Quantitative Polymerase Chain Reactions (qPCR) for species identification were performed in 15µl reactions containing 7.5µl Quantitect Multiplex NO-Rox Master Mix (Qiagen), 0.75µl primer-probe mix (primer concentration 8µM each, probe concentration 4µM), 0.3µl TaqMan 50X Exogenous IPC DNA (Applied Biosystems), 0.6µl TaqMan 10X Exogenous Internal Positive Control (IPC) Block (Applied Biosystems), and 2.85µl diH2O. Samples were run in triplicate on a 7500 Real-Time PCR System (Applied Biosystems) using standard dilutions made from tissue DNA extracts for each species. Amplification curves were visualized using the 7500 RT PCR System software. Negative controls were included in each step downstream to identify cross-contamination. bobcat positive samples were identified and DNA was used in subsequent steps.