Hot start taq mix

We performed DNA extraction from all field-collected scats using already established approaches described in Modi et al.^{27 (link)}. In brief, we either swabbed twice (samples with no dust) or scraped (samples covered with dust) the top layer of the samples with sterile swabs or blade, respectively. They were lysed overnight in a lysis buffer at 56 °C, and extraction was performed following QIAamp DNA Tissue Kit (Qiagen Inc, Hilden, Germany) protocol. Final elution was performed twice in 100 μl of 1X TE buffer, and the DNA was stored at − 20 °C for long-term use.
We conducted molecular species identification using dhole-specific mitochondrial DholespID-F/R primers described in Modi et al.^{27 (link)}. PCR reactions were performed in 10 µL volumes with 4 µL of hot-start taq mix (Qiagen Inc, Hilden, Germany), 4 µM BSA, 0.5 µM of primer mix and 3 µL of DNA extract. PCR conditions included an initial denaturation (95 °C for 15 min); 50 cycles of denaturation (94 °C for 30 s), annealing (50 °C for 30 s) and extension (72 °C for 35 s); followed by a final extension (72 °C for 10 min). Negative and extraction controls were included to monitor contaminations. Species ascertainment was done through visualization of dhole-specific bands (236 bp) in 2% agarose gel. All the experiments were conducted in Conservation Genetics Lab in Wildlife Institute of India, Dehradun.

NCCR sequences were amplified by nested PCR using outer primer pair 1 and inner primer pair 2 (S2 Table). HotStartTaq® mix (QIAGEN) was used to amplify 1 to 5 ng of DNA with 400 nM of pair 1 primers using these cycle conditions: a denaturation/activation step of 15 min at 95°C and then 45 cycles of amplification (94°C for 45 s, 55°C for 30 s, 72°C for 45 s). For CSFs from patients 3 and 4, 1 µl of amplicon was used to perform a second PCR with the inner primer pair 2 in the same conditions except that the 35 cycles of amplification were performed with a hybridization step at 52°C. DNA sequencing of PCR products was performed by automatic DNA sequencer (3130 Genetic Analyzer, Applied Biosystems®), according to the manufacturer’s specifications. The sequences were aligned to the CY at-NCCR [12 (link)] with the program BioEdit and divided into sequence sections a, b, c, d, e, and f consisting of 25, 23, 55, 66, 18 and 69 bp, respectively (Fig 1).