Characterization of target nuclear DNA from faeces reduces technical issues associated with the assumptions of low-quality and quantity template

Abstract

Faecal material has increasingly become an important non-invasive source of DNA for wildlife population genetics. However, DNA from faecal sources can have issues associated with quantity (lowtemplate and/or low target-to-total DNA ratio) and quality (degradation and/or low DNA-to-inhibitor ratio). A number of studies utilizing faecal material assume and compensate for the above properties with minimal characterization of quantity or quality of target DNA, which can unnecessarily increase the risk of downstream technical problems. Here, we present a protocol which quantifies faecal DNA using a two step approach: (1) estimating total DNA concentration using a Picogreen TM fluorescence assay and (2) estimating target nuclear DNA concentration by comparing amplification products of field samples at suspected concentrations to those of control DNA at known concentrations. We applied this protocol to faecal material collected in the field from two species: woodland caribou (Rangifer tarandus) and swift fox (Vulpes velox). Total DNA estimates ranged from 6.5 ng/ll to 28.6 ng/ll (X = 16.2 ng/ll) for the caribou extracts and 1.0-26.1 ng/ll (X = 7.5 ng/ll) for the swift fox extracts. Our results showed high concordance between total and target DNA estimates from woodland caribou faecal extracts, with only 10% of the samples showing relatively lower target-to-total DNA ratios. In contrast, DNA extracts from swift fox scat exhibited low target DNA yields, with only 38% (19 of 50) of the samples showing comparative target DNA amplification of at least 0.1 ng. With this information, we were able to estimate the amount of target DNA entered into PCR amplifications, and identify samples having target DNA below a lower threshold of 0.2 ng and requiring modification to genotyping protocols such as multiple tube amplification. Our results here also show that this approach can easily be adapted to other species where faeces are the primary source of DNA template.

Comparative woodland caribou population surveys in Slate Islands Provincial Park, Ontario

“…One must also consider timing restrictions with each survey technique. Both FLIR and ground surveys are best conducted when deciduous vegetation has lost its leaves and DNA extraction for genetic analysis produces better results with winter collections of faecal pellets (Ball et al, 2007). The caribou population estimate from the FLIR survey represents a minimal value that may have been limited by the rugged landscape of the Slate Islands and possibly dense conifer forest cover; subsequent FLIR surveys for moose and caribou in a conifer-dominated landscape on the mainland north of the Slate Islands, however, indicated this forest type does not severely limit detection (A. Rodgers, unpubl.…”

“…Laboratory analyses Caribou DNA was extracted from faecal samples using the methods of Ball et al (2007). DNA was amplified using 9 polymorphic, microsatellite markers (Rt6, Rt7, Rt24, Rt30 (Wilson et al, 1997); Map2C, BM848 (Moore et al, 1992); BM888, RT5 (McLoughlin et al, 2004); BMS1788 (Cronin et al, 2005).…”

Sex Determination of Sika Deer (Cervus nippon yesoensis) Using Nested PCR from Feces Collected in the Field

“…As for factors related to the DNA condition, climate including temperature, humidity and rainfall, as well as diet or elapsed time from defecation are suggested [3,17,24]. For feces in a low DNA condition, repeating fecal DNA extraction or PCR amplification will be needed due to allelic dropout and false alleles resulting from DNA degradation [2,11,25]. Therefore, a pilot study for determining a suitable strategy was recommended.…”

“…In this situation, fecal samples that can be collected noninvasively are simply counted instead of biomaterials and studies using fecal samples have been conducted in many species [2,5,11,23,25,30]. To use fecal samples for further analysis beyond genotyping, sex information will be essential.…”

Does connectivity exist for remnant boreal caribou (Rangifer tarandus caribou) along the Lake Superior Coastal Range? Options for landscape restoration

“…DNA sample concentrations were determined by PicoGreen and samples were normalized to 2.5 ng/ul to ensure reliable amplification of samples. Extracted DNA was amplified at nine microsatellite loci following Ball et al, (2007Ball et al, ( , 2010. Amplification reactions contained: 1x PCR buffer; 2.0 mM MgCl; 0.2 μg/ml of BSA; 0.4 μM of each primer pair; 0.2 μM of each dinucleotide triphosphate; 0.5 units of Taq polymerase (Invitrogen Life Technologies); and 5 ng of DNA template.…”