Evaluation of Fecal Storage and DNA Extraction Methods in Wild Boar (Sus scrofa)

Kołodziej, Karolina; Nikolov, Ivan; Schulz, H. K.; Theissinger, Kathrin; Schulz, Ralf

doi:10.1007/s10528-013-9573-1

“…Additionally, studying longitudinal changes in microbiota has paved the way for developing microbiome tools to study unique signatures and providing, at least in theory, the means to locate people in space and time in forensic science (Kim, M, Zorraquino & Tagkopoulos, 2015;Fornaciari, 2017). A subset of our study investigates longitudinal changes in However, there is little consensus on the optimal buffer and temperature condition for bacterial preservation (Nechvatal et al, 2008;Cardona et al, 2013;Kolodziej et al, 2013;McKain et al, 2013;Fliegerova et al, 2014;Hale et al, 2015;Song et al, 2016;Hale et al, 2016;Metzler-Zebeli et al, 2016). Furthermore, a limited number of studies have evaluated the combination of these chemical buffers with temperature reduction to optimize DNA preservation.…”

Section: Discussionmentioning

confidence: 96%

Peer Review #1 of "Effects of preservation method on canine (Canis lupus familiaris) fecal microbiota (v0.1)"

Hale¹

2018

0

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Studies involving gut microbiome analysis play an increasing role in the evaluation of health and disease in humans and animals alike. Fecal sampling methods for DNA preservation in laboratory, clinical, and field settings can greatly influence inferences of microbial composition and diversity, but are often inconsistent and under-investigated between studies. Many laboratories have utilized either temperature control or preservation buffers for optimization of DNA preservation, but few studies have evaluated the effects of combining both methods to preserve fecal microbiota. To determine the optimal method for fecal DNA preservation, we collected fecal samples from one canine donor and stored aliquots in RNAlater, 70% ethanol, 50:50 glycerol:PBS, or without buffer at 25°C, 4°C, and-80°C. Fecal DNA was extracted, quantified, and 16S rRNA gene analysis performed on days 0, 7, 14, and 56 to evaluate changes in DNA concentration, purity, and bacterial diversity and composition over time. We detected overall effects on bacterial community of storage buffer (F-value= 6.87, DF= 3, P<0.001), storage temperature (F-value=1.77, DF= 3, P=0.037), and duration of sample storage (F-value=3.68, DF= 3, P<0.001). Changes in bacterial composition were observed in samples stored in-80°C without buffer, a commonly used method for fecal DNA storage, suggesting that simply freezing samples may be suboptimal for bacterial analysis. Fecal preservation with 70% ethanol and RNAlater closely resembled that of fresh samples, though RNAlater yielded significantly lower DNA concentrations (DF=8.57, P<0.001). Although bacterial composition varied with temperature and buffer storage, 70% ethanol was the best method for preserving bacterial DNA in canine feces, yielding the highest DNA concentration and minimal changes in bacterial diversity and composition. The differences observed between samples highlight the need to consider optimized post-collection methods in microbiome research.

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“…Using DNA data as a component of recapture instead of physical recapture for estimating population densities in a CMR or SCR environment also provides results with high quality and accuracy (Ebert et al, 2012;Kery et al, 2011). Compared to DNA-based sampling methods like feces sampling (Bellemain et al, 2005;Ebert et al, 2012;Kolodziej et al, 2013), genotyping of the hunting bag requires less time and sampling effort and provides a steady and faster genotyping success due to superior DNA quality of muscle tissue. Additionally, this method gives a surplus of information regarding the population.…”

Section: Discussionmentioning

confidence: 99%

“…Counting fecal pellets can provide reliable results (Ferretti, Fattorini, Sforzi, & Pisani, 2016;Plhal, Kamler, & Homolka, 2014) but in some cases can lead to over-or underestimated densities compared to other methods (Barnes, 2001;Pfeffer et al, 2018). The choice of appropriate transects, often unavoidably high staff expenses, aggravated long-term storage for later DNA analysis and DNA amplification, and weather-dependent feces quality can make feces counts and genetic analyze of the fecal pellets a protracted and time-consuming process with an uncertain outcome (Barnes, 2001;Bellemain et al, 2005;Kolodziej, Nikolov, Schulz, Theissinger, & Schulz, 2013;Soto-Calderon et al, 2009).…”

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confidence: 99%

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Population size estimates based on the frequency of genetically assigned parent–offspring pairs within a subsample

Müller

¹

,

Mercker

²

,

Brün

³

2020

Ecology and Evolution

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Estimating population density as precise as possible is a key premise for managing wild animal species. This can be a challenging task if the species in question is elusive or, due to high quantities, hard to count. We present a new, mathematically derived estimator for population size, where the estimation is based solely on the frequency of genetically assigned parent–offspring pairs within a subsample of an ungulate population. By use of molecular markers like microsatellites, the number of these parent–offspring pairs can be determined. The study's aim was to clarify whether a classical capture–mark–recapture (CMR) method can be adapted or extended by this genetic element to a genetic‐based capture–mark–recapture (g‐CMR). We numerically validate the presented estimator (and corresponding variance estimates) and provide the R‐code for the computation of estimates of population size including confidence intervals. The presented method provides a new framework to precisely estimate population size based on the genetic analysis of a one‐time subsample. This is especially of value where traditional CMR methods or other DNA‐based (fecal or hair) capture–recapture methods fail or are too difficult to apply. The DNA source used is basically irrelevant, but in the present case the sampling of an annual hunting bag is to serve as data basis. In addition to the high quality of muscle tissue samples, hunting bags provide additional and essential information for wildlife management practices, such as age, weight, or sex. In cases where a g‐CMR method is ecologically and hunting‐wise appropriate, it enables a wide applicability, also through its species‐independent use.

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“…However, there is little consensus on the optimal buffer and temperature condition for bacterial preservation (Nechvatal et al, 2008;Cardona et al, 2013;Kolodziej et al, 2013;McKain et al, 2013;Fliegerova et al, 2014;Hale et al, 2015;Song et al, 2016;Hale et al, 2016;Metzler-Zebeli et al, 2016). Furthermore, a limited number of studies have evaluated the combination of these chemical buffers with temperature reduction to optimize DNA preservation.…”

Section: Discussionmentioning

confidence: 99%

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Supplemental Information 4: Fig S4. Species Evenness of All Samples by Buffer, Temperature, Day, and Their Interactions

0

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Studies involving gut microbiome analysis play an increasing role in the evaluation of health and disease in humans and animals alike. Fecal sampling methods for DNA preservation in laboratory, clinical, and field settings can greatly influence inferences of microbial composition and diversity, but are often inconsistent and under-investigated between studies. Many laboratories have utilized either temperature control or preservation buffers for optimization of DNA preservation, but few studies have evaluated the effects of combining both methods to preserve fecal microbiota. To determine the optimal method for fecal DNA preservation, we collected fecal samples from one canine donor and stored aliquots in RNAlater, 70% ethanol, 50:50 glycerol:PBS, or without buffer at 25°C, 4°C, and-80°C. Fecal DNA was extracted, quantified, and 16S rRNA gene analysis performed on days 0, 7, 14, and 56 to evaluate changes in DNA concentration, purity, and bacterial diversity and composition over time. We detected overall effects on bacterial community of storage buffer (F-value= 6.87, DF= 3, P<0.001), storage temperature (F-value=1.77, DF= 3, P=0.037), and duration of sample storage (F-value=3.68, DF= 3, P<0.001). Changes in bacterial composition were observed in samples stored in-80°C without buffer, a commonly used method for fecal DNA storage, suggesting that simply freezing samples may be suboptimal for bacterial analysis. Fecal preservation with 70% ethanol and RNAlater closely resembled that of fresh samples, though RNAlater yielded significantly lower DNA concentrations (DF=8.57, P<0.001). Although bacterial composition varied with temperature and buffer storage, 70% ethanol was the best method for preserving bacterial DNA in canine feces, yielding the highest DNA concentration and minimal changes in bacterial diversity and composition. The differences observed between samples highlight the need to consider optimized post-collection methods in microbiome research.

show abstract

Evaluation of Fecal Storage and DNA Extraction Methods in Wild Boar (Sus scrofa)

Cited by 6 publications

References 22 publications

Peer Review #1 of "Effects of preservation method on canine (Canis lupus familiaris) fecal microbiota (v0.1)"

Peer Review #1 of "Effects of preservation method on canine (Canis lupus familiaris) fecal microbiota (v0.1)"

Population size estimates based on the frequency of genetically assigned parent–offspring pairs within a subsample

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