Frozen swab and scat samples provide sufficient DNA for mtDNA sequencing
Mitochondrial (cytB region) amplification success was high with 100% of blood samples (5/5) and 71% (43/63) of scat samples producing clear bands of the expected size (~420 bp, Table 1). Amplification success was greater for DNA isolated from scat swabs (81%, 21/26) than from DNA isolated from scat material taken from whole scats (59%, 22/37; Table 1). This is probably because swab samples were first collected from many of the scats which comprised the whole scat samples; and suggests that compared to the scat interior, DNA quality may be greater when sampled from the scat surface.
Scat quality assessments made at the time of sample collection were available for approximately 70% (28/40) of the scat samples used in this study, with 22% (9/40) and 49% (19/40) being regarded as good or poor quality, respectively. For scats that were categorised as good at the time of collection, swab DNA amplified 100% (6/6) of samples, while 63% (5/8) of the whole scat samples resulted in successful amplification of isolated DNA (Table 1). Scats that were classified as poor quality performed worse than good quality scats, however, amplification was still achieved for 78% (11/14) of DNA samples isolated from swabs, and 47% (9/19) samples isolated from scat material (Table 1).
A subset of 37 samples that successfully amplified a segment of thecytochrome b gene (blood n =5, scat material n =12 and scat swab n =20) were selected for Sanger sequencing of which 65% (24/37) samples produced readable data. Sequencing success was, as expected, high for DNA isolated from blood, with all five samples producing good sequence data. For scats, 59% (19/32) of samples produced reliable DNA sequence data, where success was similar for both scat material (58%, 7/12) and scat swabs (60%, 12/20; Table 1). Overall, scats classed as being in good condition provided sequence data more reliably (75%, 6/8) than those classed as being in poor condition (11%, 2/18; Table 1). DNA isolated from scat material and sent for sequencing had an associated scat quality assessment for only five samples (one classed as being in good condition and four in poor condition) with all of these samples failing to produce quality sequence data (Table 1). DNA isolated from scat swabs described as being in good condition produced better sequencing results (100%, 6/6) than those described as being in poor condition (20%, 2/10). These data indicate that a visual assessment score of scats could be useful for identifying pangolin scat samples that would have a greater chance of providing DNA of sufficient quality for genetic analysis.