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.