Sample origin and DNA isolation
Both scat and blood samples, previously collected between 2017 and 2020 from two broad geographic regions in Nepal were provided as stored, frozen samples by Nepal’s National Trust for Nature Conservation (NTNC). Scat samples originated from east (Taplejung, n =23) and central (Kathmandu, n =4; Chitwan, n =16) Nepal (Fig. 1) and were mostly collected in 2017 as part of another study (Kim 2021). Two additional samples were from an unknown location in Nepal. Scat samples were collected whole, and the surface of the scat swabbed at the time of collection (n =26). Swabs and whole scats (n =37,) were both stored frozen (–20°C) for up to 3 years. Samples were collected from 40 putative individuals and included 23 paired scat and swab samples, 14 scats without a paired swab and 3 swabs without a paired scat. Some of the scat samples had been visually assessed at the time of collection as being in either ‘good’ or ‘poor’ condition; intact scats appearing fresh (e.g. signs of moisture) were classed as good quality while those appearing dry, degraded or mouldy were classified as poor quality. NTNC also provided access to stored blood samples (n =5) which had been taken from pangolins rescued within the Kathmandu (n =4) and Chitwan (n =1) areas and stored at –20°C for approximately 1.5 years.
DNA was isolated from blood samples using the DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer’s instructions, except DNA was eluted in 50 μL. Swab samples were prepared for DNA isolation by allowing the samples to thaw at room temperature and then vortexing the tubes for approximately 3 minutes, ensuring that material adhering to the swab was released into the liquid. Swabs were removed from each tube followed by centrifugation at 10,000 rpm for 10 minutes. A pipette was used to remove most of the supernatant, leaving the pellet undisturbed. Samples were then vortexed and DNA isolated using the QIAamp DNA Stool Mini Kit (Qiagen). DNA was isolated following the manufacturers protocol except that the sample–buffer ASL mixture was incubated for one hour at 35°C to ensure that a homogenous solution was obtained. DNA was eluted using two separate aliquots of 50 μL and combined.
Frozen whole scat samples were thawed at room temperature. Pieces of scat material were transferred from each whole scat to a 2 mL microcentrifuge tube, which was loosely filled to approximately half of the containers volume (this equated to 200 – 400 mg of scat material). Scat material was then washed by addition of 1.5 mL of phosphate buffered saline (PBS) to each tube, vortexed thoroughly and placed on a tube rotator for 10 minutes. The liquid was then transferred to a new tube and centrifuged at 7,500 rpm for 5 minutes. Most of the supernatant was discarded and DNA was then extracted using the QIAamp DNA Stool Mini Kit (Qiagen) with a one-hour lysis incubation.