A Paired Comparison of Scat-collecting versus Scat-swabbing Methods for Noninvasive
Recovery of Mesocarnivore DNA from an Arid Environment
Miles, K.A, Holtz, M.N., Lounsberry Z.T., Sacks, B.N.
Supporting Information 2. Comparison of 3 additional swab-storage and processing
approaches to the 2 methods reported in the main paper.
We conducted 3 additional swab processing approaches (swab variations B–D), in addition to the
direct-ethanol and swab methods described in the main text, using 5 scats. For methods
corresponding to the direct-ethanol approach and primary swabbing approach, see main text. The
3 additional swab protocols all involved submerging swabs in 1.5 mL of 95–100% ethanol inside
2-mL microcentrifuge tubes instead of dry-storage in manila coin envelopes (as in the main text).
For variation B, we carried samples in a backpack for 15 minutes to simulate the jostling
associated with typical field transportation, then removed swabs and incubated them for 10
minutes at 70° C to evaporate ethanol before transferring them to digestion tubes (700 µL of
Phosphate Buffered Saline, 200 µL of Buffer AL, and 25 µL of Proteinase K). For variation C,
instead of jostling in a backpack, we vortexed the sample for 1 minute, again, followed by
removing the swab and incubating for 10 minutes at 70° C to evaporate ethanol before
transferring them to digestion tubes. For variation D, we utilized the residual ethanol remaining
in the vortexed tube (corresponding to the removed swab in variation C). We then evaporated the
ethanol using a vacuum centrifuge for 30 minutes and (after removing from the centrifuge) added
the digestion reagents directly to the tube. To remove swabs from the digestion tubes in
variations B and C, we vortexed samples for 5–10 seconds, centrifuged them for 1 minute at
14,000 revolutions/minute, and pressed swabs against the sides of the tubes to remove any
remaining liquid before discarding. All subsequent steps were the same. We extracted DNA
using a DNeasy Blood and Tissue Kit (Qiagen, Toronto, ON, Canada) following manufacturer’s
instructions, except that we eluted samples in 100 uL of buffer (EB).
We tested mitochondrial sequencing success, microsatellite genotyping success, and
canine DNA yield from these extracts (see Methods in main text). We obtained positive speciestyping results (mitochondrial amplification and sequencing) for all 5 samples with the directethanol approach, which revealed 4 red fox samples and 1 house cat sample. All 4 of the swab
extracts also produced positive species-typing results for the 4 red fox samples, but none of them
identified the house cat sample. Using the samples corresponding to the 4 red fox scats, we
amplified ≥5 of the 7 microsatellite loci in all 20 extracts (i.e., 5 methods × 4 scats).
However, an analysis of variance indicated that the DNA content differed significantly among
methods (F4,15 = 3.92, P = 0.022). A Fisher’s LSD post hoc test indicated significantly higher
concentrations in the direct-ethanol extracts than in all 4 swab-method extracts (P ≤ 0.012),
which were uniformly low and did not differ significantly from one another (P ≥ 0.692; S2
Table 1, S2 Fig. 1).
S2 Table 1. Comparison of DNA yields (ng/µL) based on qPCR for canid-specific DNA among
5 methods employed on 4 different red fox scats.
Scat
Direct-ethanol
Swab (dry)
Swab var. B
Swab var. C
Swab var. D
Red fox 1
16.420
0.015
0.027
0.006
0.009
Red fox 2
5.530
0.013
0.006
0.018
0.004
Red fox 3
0.694
0.003
0.020
0.019
0.051
Red fox 4
0.162
1.050
0.162
0.088
0.021
DNA concentration [ ln (ng/ul + 1) ]
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Method
S2 Figure 1. Comparison of average DNA concentrations, Loge[(ng/µL) + 1], among extracts
corresponding to 5 methods of sample collection and storage. The error bars represent one
standard error.