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.