Impact of two field preservation methods on genotyping success of feces

Introduction

An important source of data for many molecular ecology and conservation genetic studies is feces collected from animals in the wild. Feces collection enables the genetic study of elusive and endangered animals without disturbing them, through a non-invasive sampling (Taberlet et al., 1999). The analysis of fecal DNA can document the presence of species or the identification of individuals (e.g., Kohn et al., 1999; Mumma et al., 2015; Stenglein et al., 2010a; Stenglein et al., 2010b) and yields insights into spatial ecology and social behavior (Forcina et al., 2019; Goldberg et al., 2020; Ibáñez et al., 2016). Although this particular type of sample is a valuable window into the ecology, behavior and population biology of wild species, it also has some disadvantages. The DNA extracted from feces is generally degraded, has low concentration, and is mixed with many unknown inhibitors and DNA from other species (Deagle, Eveson & Jarman, 2006; Kohn & Wayne, 1997). These features create logistical hurdles and complicate the high throughput processing of DNA from feces.

Fecal DNA is extensively used to amplify fragments of mitochondrial DNA for host identification (barcoding) and genotyping with nuclear microsatellites (e.g., Galan, Pagès & Cosson, 2012; Gil-Sánchez et al., 2020; Muñoz Fuentes et al., 2009). These techniques have been heavily dependent on gel electrophoresis for separating fragments either for Sanger sequencing or genotyping of microsatellite loci by length polymorphism. The equipment necessary to perform these analyses can not process very many samples at a time (generally up to 96) and are becoming obsolete. However, novel approaches have been developed in recent years that involve the use of massive sequencing approaches that can revitalize the use of these genetic markers by scaling up and solving some of the main problems that microsatellite typing had as compared to single-nucleotide polymorphism (SNP) typing, such as low throughput and automation, as well as lack of transferability across laboratories, or even between projects within the same laboratory (De Barba et al., 2017; Guichoux et al., 2011; Zhan et al., 2017). The optimal use of resources requires every step of the process, from experimental set-up and sample collection in the field (Sarabia et al., 2020) to genotyping (Salado et al., 2021), to be revised and optimized to be compatible with genomics technology. Here we focus on the impact of field preservation of samples on genotyping success.

The most common field preservation method is to put the fecal sample (whole or fragments) in 70–100% ethanol to ship it to the lab at room temperature (de Oliveira, Gonçalves & Galetti Jr, 2025; Panasci et al., 2011). Dry preservation (for example with silica) has also been shown to preserve DNA (Frantzenm Ma et al., 1998), but is less practical in humid environments. Working with ethanol has some disadvantages. First, it is volatile, so the concentration may change substantially if the tube is left open for a while or not completely sealed, resulting in a much lower ethanol concentration (worse for DNA preservation). If several samples are collected, the volume of ethanol can become a problem for shipping because it is flammable. Finally, once in the lab, samples preserved in ethanol need to be completely dried before processing because even a small amount of ethanol will denature enzymes, such as proteinase K. This drying process takes time and may increase the risk of contamination while tubes are left open in the lab. Lastly, whether the sample was kept dry since collection or dried in the lab, handling dry feces increases exposure to air-borne particles -including parasite eggs- for laboratory personnel (Sánchez Thevenet et al., 2003). A potential solution to these problems would be the use of nucleic acid preservation (NAP) buffer (Camacho-Sanchez et al., 2013). Similar to RNAlater^® (Qiagen), NAP buffer is a salt solution (see Methods for composition details) that stabilizes and protects DNA and RNA from degradation by permeating the tissue and inhibiting enzymatic activity. NAP buffer is non-hazardous and non-flammable, making it easy to ship. Also, samples preserved in NAP do not need to be dried before DNA extraction, which shortens processing time, decreases the risk of contamination, and reduces the risk to the technician of inhaling disease vectors.

NAP buffer preserves DNA quality and quantity slightly better than 95% ethanol for high quality tissue samples, but high molecular weight DNA is preserved in both conditions (Camacho-Sanchez et al., 2013). However, samples such as feces, which contain a wide variety of contaminants, may provide other challenges when preserving DNA. Ethanol is the most common field preservation method for fecal samples (Panasci et al., 2011), but the impact of these two preservation methods on genotyping success of fecal samples has not been previously assessed using next-generation sequencing data. Here we evaluate the quality of genotypes from fecal samples preserved in ethanol and in NAP buffer. We hypothesize that both preservation methods will perform similarly and no difference in the quality of genotypes of fecal samples will be found.

Materials and Methods

Materials

Twelve feces presumed to correspond to Iberian gray wolves (Canis lupus) were collected in northern Spain in July 2020 (Table S1). In the field, a 5-cm fragment (approximately 20 g, wet weight) was taken from each feces and submerged in 30 mL 96% ethanol in a 50 mL Falcon tube (0.67:1, sample:solution), and another fragment of the same size in a tube with the same amount of NAP buffer (Fig. 1). The NAP buffer consisted of 0.019 M Ethylenediaminetetra-acetic acid (EDTA) disodium salt dihydrate, 0.018 M sodium citrate trisodium salt dihydrate, and 3.8 M ammonium sulphate, and was adjusted to pH 5.2 with H₂SO₄; (see Camacho-Sanchez et al., 2013 for the full protocol). All samples were kept at ambient temperature for three weeks before shipping to the lab. Feces were received at the end of July 2020 and stored at −80 °C until processing in the lab in February 2021.

Statistical analyses

We statistically compared the effect of the two preservation media over AS and GS by fitting two generalized linear mixed models (GLMM) under a negative binomial family (nbinom2) with the function glmmTMB() from the package glmmTMB (Brooks et al., 2017). We used PCR replicate for each locus and feces as sample unit in both models, and the two levels for the response factors AS and GS were failure (0) or success (1) (Table S3). As we observed differences in the total number of reads assigned to a locus between the two preservation media (see Results, Fig. 2), we added coverage (i.e., number of reads per PCR replicate/locus/feces) as a fixed factor in the models (Table S3). We scaled coverage, by using the scale() function, which standardized the variable by subtracting the mean and dividing by the standard deviation. We also controlled the effect of the differences among feces and locus by including them and their interaction as random factors in the models. Normality of residuals was checked by visual exam of scatterplots. Significance was evaluated with a Chi-square test using the Anova() function available in the R car package (Fox & Weisberg, 2018). Statistical analyses were performed with R version 4.4.2 (R Core Team, 2024) by using RStudio version 2024.12.0 (Posit team, 2024).

Results

Amplification and genotyping success

We obtained 184 genotypes (109 heterozygotes, 75 homozygotes) out of 384 total possible genotypes (12 feces × 32 loci) to be used as reference (Table S4). From those without a genotype, 48 were considered ‘ambiguous’ following our genotyping criteria (see Methods). For downstream analyses, we only considered the 184 reference genotypes. All fecal samples yielded more than ten reference genotypes (out of 32), except for three (feces #1, 6 and 11; Fig. S2). GLMM analyses showed that samples in ethanol had a higher Amplification success (AS = 0.62, Chisq₁ = 16.88, p < 0.001), and also a higher Genotyping success (GS = 0.87, Chisq₁ = 25.28, p < 0.001) than those preserved in NAP (AS = 0.52; GS = 0.77) due primarily to a higher rate of allelic dropout in the NAP preserved samples (ADO NAP = 0.27; ADO Ethanol = 0.17) (Table 1; Fig. 3). Coverage (i.e., number of reads per PCR replicate/locus/feces) showed a significant effect only over amplification success (AS, Chisq₁ = 14.44, p < 0.001), meaning that a higher coverage leads to obtaining more PCR replicates with a genotype, but not necessarily with a genotype that is correct.

Number of replicates

Both the amplification and the genotyping success were higher for samples preserved in ethanol. To assess the potential impact of these differences on the cost and reliability of a feces genotyping project, we calculated how they affected the number of replicates needed to genotype individual feces for different numbers of loci. Results showed that the number of replicates necessary to have high quality genotypes was lower for samples preserved in ethanol. For example, for samples preserved in ethanol, seven replicates would be needed for obtaining a correct genotype with a probability higher than 95% for 20 of the 32 microsatellites, while samples preserved in NAP would require nine replicates (Fig. 4).

Discussion

The equipment, materials and software necessary for “traditional” genotyping of microsatellite loci are very quickly becoming obsolete as the industry moves towards genomics technologies. However, microsatellite loci can still be very informative in some studies (Cueva et al., 2024; Cullingham et al., 2023; Karamanlidis et al., 2021; Riquet et al., 2021), and adapting microsatellite typing protocols to next generation sequencing technologies can greatly facilitate their use (De Barba et al., 2017; Lanner et al., 2021). Experiments need to be carefully planned because one characteristic of “next generation” technologies is that they generate huge amounts of data per run, so larger units of work need to be planned at a time. The careful planning of each step can have a large impact on the quality of the final dataset (Brandariz-Fontes et al., 2015).

Various steps in the process of genotyping feces, from field collection (Sarabia et al., 2020; Scholz et al., 2024; Van Cise et al., 2024), to lab analysis (Sarabia et al., 2020), to bioinformatics (De Barba et al., 2017; Salado et al., 2021), have previously been examined to improve compatibility with high-throughput workflows. Another important step that could be optimized is preservation of samples in the field for transport to the lab. A review of the literature in Web of Science (keyword strings TS= ((noninvasive OR non-invasive OR “non invasive” OR feces OR fecal OR faecal OR faeces OR scat OR saliva OR hair OR environmental DNA OR eDNA) AND (genetic* OR molecular* OR DNA) AND (species OR wildlife) AND (microsatellite OR STR OR short-tandem repeats) AND (high throughput OR HTS OR next-generation OR next-generation OR NGS) AND (amplification success OR genotyping success))) returned 26 papers, of which only four both reported how fecal samples were preserved in the field and then were genotyped with next generation sequencing tools (Barbian et al., 2018; De Barba et al., 2017; Salado et al., 2021; Sarabia et al., 2020). These papers employed different protocols on different sample types from different species in a variety of habitats and yielded highly variable results (Table S6). The clearest result is that few papers describe their collection methods clearly, but these data would be an important addition to the literature so that future meta-analyses could be performed to better understand DNA preservation and degradation under practically important conditions.

In this study, we hypothesized that both preservation methods should preserve DNA similarly well, however we found that there was a difference between amplification and genotyping success between fecal samples preserved in ethanol and those preserved in NAP buffer. Those preserved in ethanol had a lower rate of allelic dropout and would require fewer replicates to obtain reliable genotypes at multilocus genotypes. However, the number of replicates will also depend on the research question, the specific panel of loci, and the sample type. Our estimated number of replicates are panel-specific. Since the difference between the two preservation methods in the number of replicates required to reach confidence thresholds per locus is not large, it could be relevant to also consider other benefits or disadvantages of the preservation methods when designing an experiment. Other potentially relevant characteristics include safety and processing time. NAP buffer is safe to ship because it is not flammable, and also increases safety of the lab personnel by enabling the feces to be extracted directly without drying. This can also reduce handling time, and thus overall lab processing time.

Here we have focused solely on preservation for amplification of DNA markers, but similarly preserved samples could be used for a variety of other molecular analyses as well, and ethanol and NAP could be better or worse for those other analyses. RNA, for example, does not preserve in ethanol but should preserve in NAP (Camacho-Sanchez et al., 2013). Faecal and environmental microbiota also preserve well in NAP (Menke et al., 2017; Ward et al., 2023). Different preservation methods also impact stable isotopes differently (Barrow, Bjorndal & Reich, 2008). Since the same feces may be used for a variety of different analyses (Gil-Sánchez et al., 2020), the appropriateness of preservation for the other analyses should also be taken into account.

Additionally, our results showed that there was a large variance in the amplification success for the different microsatellite loci (Fig. 2). All of the loci included here have previously been used by our group with “traditional” microsatellite analyses involving size separation in polyacrylamide gel (Hailer & Leonard, 2008; Koblmüller et al., 2009; Musiani et al., 2007). Some loci that worked well with traditional methods did not work well or at all with next generation sequencing, as Salado et al. (2021) also found with a larger multiplex microsatellite panel (46 loci), that included all the microsatellite loci used in this study. For this panel, Salado et al. (2021) recommended a coverage higher than 100 reads per PCR replicate/locus/feces using the same bioinformatic genotyping method as this study. De Barba et al. (2017) designed a new 13-microsatellite panel specifically for the analysis of degraded DNA samples with the high-throughput sequencing approach in brown bear (Ursus arctos) and obtained an improvement of genotyping success (84%) and cost reduction compared to capillary electrophoresis. Determining which characters make loci more appropriate for high throughput sequencing will be important in the planning and optimization of projects including microsatellite genotyping in the future.

We observed differences in amplification and genotyping success across fecal samples (Fig. 2, Fig. S2). An interaction between preservation methods and diet content has also been reported to impact both amplification and genotyping success in fecal DNA using traditional genotyping, specifically for the probability of false alleles (Panasci et al., 2011). Ethanol preservation has been suggested for scats of obligate carnivores and of facultative carnivores with a diet consisting primarily of mammals, while salt-solution-based buffers (e.g., dimethyl sulfoxide saline solution, DET buffer- composed by 20% DMSO, 0.25 M EDTA, 100 mM Tris, pH 7.5 and NaCl to saturation; Seutin, White & Boag, 1991) are suggested for preservation for scats of animals with a diet consisting of plant-derived foods (Panasci et al., 2011). Our wolf feces contained mainly mammals, although some included plant material, as wolves sometimes swallowed them accidentally with other food or for curative purposes (Pezzo, Parigi & Fico, 2003). However, our sample size is too limited (n = 12 feces) to find a trend between fecal content and preservation methods. Future studies could evaluate the interaction between preservation methods and fecal content and how this impacts the amplification and genotyping success in feces using genotyping based on high throughput data.

Conclusions

Ethanol is better at preserving feces for genotyping microsatellites with Illumina sequencing, but the difference between preservation methods does not result in dramatic differences in the number of replicates required to generate high quality genotypes. Thus, other considerations such as logistics and safety should be taken into account when selecting a field preservation solution. The difference in success between loci and samples is much more important than the difference in field preservation, so careful sample and locus selection will be an effective way to increase the quality of genotypes for the same effort.

Supplemental Information