Background Non-invasively collected samples allow a variety of genetic studies on endangered and elusive species. However due to low amplification success and high genotyping error rates fewer samples can be identified up to the individual level. Number of PCRs needed to obtain reliable genotypes also noticeably increase. Methods We developed a quantitative PCR assay to measure and grade amplifiable nuclear DNA in feline faecal extracts. We determined DNA degradation in experimentally aged faecal samples and tested a suite of pre-PCR protocols to considerably improve DNA retrieval. Results Average DNA concentrations of Grade I, II and III extracts were 982pg/μl, 9.5pg/μl and 0.4pg/μl respectively. Nearly 10% of extracts had no amplifiable DNA. Microsatellite PCR success and allelic dropout rates were 92% and 1.5% in Grade I, 79% and 5% in Grade II, and 54% and 16% in Grade III respectively. Our results on experimentally aged faecal samples showed that ageing has a significant effect on quantity and quality of amplifiable DNA (p<0.001). Maximum DNA degradation occurs within 3 days of exposure to direct sunlight. DNA concentrations of Day 1 samples stored by ethanol and silica methods for a month varied significantly from fresh Day 1 extracts (p<0.1 and p<0.001). This difference was not significant when samples were preserved by two-step method (p>0.05). DNA concentrations of fresh tiger and leopard faecal extracts without addition of carrier RNA were 816.5pg/μl (±115.5) and 690.1pg/μl (±207.1), while concentrations with addition of carrier RNA were 49414.5pg/μl (±9370.6) and 20982.7pg/μl (±6835.8) respectively. Conclusions Our results indicate that carnivore faecal samples should be collected as freshly as possible, are better preserved by two-step method and should be extracted with addition of carrier RNA. We recommend quantification of template DNA as this facilitates several downstream protocols.
References
[1]
Beja-Pereira A, Oliveira R, Alves PC, Schwartz MK, Luikart G (2009) Advancing ecological understandings through technological transformations in noninvasive genetics. Mol Ecol Res 9: 1279–1301.
[2]
Waits L, Paetkau D (2005) Noninvasive genetic sampling tools for wildlife biologists: a review of applications and recommendations for accurate data collection. J Wild Manage4: 1419–1433.
[3]
Santini A, Lucchini V, Fabbri E, Randi E (2007) Ageing and environmental factors affect PCR success in wolf (Canis lupus) excremental DNA samples. Mol Ecol Notes 7: 955–961.
[4]
Murphy MA, Waits LP, Kendall KC, Wasser SK, Highbee JA, et al. (2002) An evaluation of long-term preservation methods for brown bear (Ursus arctos) faecal DNA samples. Cons Genetics 3: 435–440.
[5]
Taberlet P, Griffin S, Goossens B, Questiau S, Manceau V, et al. (1996) Reliable genotyping of samples with very low DNA quantities using PCR. Nucl Acids Res 24: 3189–3194.
[6]
Nsubuga AM, Robbins MM, Roeder AD, Morin PA, Boesch C, et al. (2004) Factors affecting the amount of genomic DNA extracted from ape faeces and the identification of an improved sample storage method. Mol Ecol 13: 2089–2094.
[7]
Morin PA, Chambers KE, Boesch C, Vigilant L (2001) Quantitative polymerase chain reaction analysis of DNA from noninvasive samples for accurate microsatellite genotyping of wild chimpanzees (Pan troglodytes verus). Mol Ecol 10: 1835–1844.
[8]
Wasser SK, Houston CS, Koehiler GM, Cadd GG (1997) Techniques for applications of faecal DNA methods to field studies of Ursids. Mol Ecol 6: 1091–1097.
[9]
Roeder AD, Archer FI, Poinar HN, Morin PA (2004) A novel method for collection and preservation of faeces for genetic studies. Mol Ecol Notes 4: 761–764.
[10]
Reed JZ, Tollit DJ, Thompson P, Amos W (1997) Molecular Scatology: the use of molecular genetic analysis to assign species, sex and individual identity to seal faeces. Mol Ecol 6: 225–234.
[11]
Kishore R, Hardy WR, Anderson VJ, Sanchez BS, Buoncristiani MR (2006) Optimization of DNA extraction from low-yield and degraded samples using the Biorobot EZ1 and Biorobot M48. J Foren Sc 51: 1055–1061.
[12]
Mondol S, Karanth KU, Kumar NS, Gopalaswamy AM, Andheria A, et al. (2009) Evaluation of non-invasive genetic sampling methods for estimating tiger population size. Biol Cons 142(10): 2350–2360.
[13]
Bhagavatula J, Singh L (2006) Genotyping faecal samples of Bengal tiger Panthera tigris tigris for population estimation: A pilot study. BMC Genetics 7: 48.
[14]
Menotti-Raymond M, David VA, Lyons LA, Schaffer AA, Tomlin JL, et al. (1999) A genetic linkage map of microsatellites of the domestic cat (Felis catus). Genomics 57: 9–23.
[15]
Arandjelovic M, Guschanski K, Schubert G, Harris TR, Thalmann O, et al. (2009) Two-step multiplex polymerase chain reaction improves the speed and accuracy of genotyping using DNA from noninvasive and museum samples. Mol Ecol Res 9: 28–36.
[16]
Piggott MP (2004) Effect of sample age and season of collection on the reliability of microsatellite genotyping of faecal DNA. Wildl Res 31: 485–493.
[17]
Deagle BE, Eveson JP, Jarman SN (2006) Quantification of damage in DNA recovered from highly degraded samples – a case study on DNA in faeces. Front Zool 3: 1–11.
[18]
Broquet T, Menard N, Petit E (2007) Noninvasive population genetics: a review of sample source, diet, fragment length and microsatellite motif effects on amplification success and genotyping error rates. Cons Gen 8: 249–260.
