Results
Our results show that the preservation solutions have a clear effect on the rate of DNA degradation. The multi-factor ANOVA showed that the variable solution had a significant effect on DNA degradation,p -value: 5.07 x 10-12 (Table 2). In addition, interactions between variables were mildly significant, suggesting interactions between temp:time and solution:temp:time ,p -values: 0.020 and 0.033 respectively. However, no significant difference was found between ANOVAs including all treatments and only including solution as a variable, p -value: 0.311. Patterns changed when samples from each solution were analyzed independently (Table 2). Samples stored in DESS showed a significant interaction between temp:time , p -value: 0.020. This was also observed when all samples were combined. DNA fragmentation for samples stored in ethanol was significantly influenced by the heat treatment, p-value: 0.007. Finally, an interaction betweenheat:temp was observed for samples stored in ethanol, p-value: 0.036.
Over time, DNA showed clear signs of degradation from samples stored in both preservation solutions (Figure 1). However, DNA from samples stored in ethanol showed significant signs of degradation after just one day. In contrast, DNA from samples stored in DESS appeared to maintain stable levels of HMW DNA fragments for up to one month following sample collection. Evidence of DNA degradation was detected in the DESS treatment after 3 months, observed by an increase of fragment sizes around 1 kbp and the frequency reduction of > 20 kbp fragments. A significant effect of heat treatment in the ethanol preserved samples could also be observed (Figure S1).
The proportion of HMW DNA for all treatment groups was < 16% (Table 3). As a proxy, samples stored for 24 hours in DESS were used as a standardized baseline because DNA was best preserved in that group. Over the first 2 weeks, samples stored in DESS had approximately 5.5 times more HMW DNA compared to the ethanol treatment (Table 3). After 3 months, samples stored in DESS still had twice the amount of HMW DNA compared to samples stored in ethanol.
Discussion
This study showed that sample preservation significantly influences the proportion and quality of extracted HWM DNA from fish samples. DESS was better at preserving DNA than ethanol under the conditions in which they were tested. Based on these findings, we conclude that DESS is best suited for preserving DNA for NGS applications. Our observations were consistent with previous studies that had included DESS in their experimental design (Dawson et al., 1998, Michaud & Foran, 2011, Seutin et al., 1991). Although these studies used different metrics to quantify DNA quality, DESS was consistently found to outperform ethanol under a range conditions. Regardless, ethanol will likely remain a popular choice for sample preservation. However, we recommend testing how well various preservation solutions perform based on the environmental conditions experienced in the field. Sample collection is the first step in any research project, which often involves a tremendous amount of effort from many people. Based on results of this study, we argue that it is worth evaluating the method of preservation to ensure samples will be yield the best data possible.
The multifactorial ANOVA suggested that solution type was the main factor contributing to the variation observed across DNA samples (Table 2). Storage temperature and time only had a significant effect when DESS was included in the analyses. This finding supports the notion that samples stored at lower temperatures degrade at lower rates, and that the rate is dependent on the storage solution. This is confirmed by the significant interaction between the variables solution:temp:time . Initial levels of DNA degradation for samples stored in ethanol were likely too high for any significant temporal patterns to be observed. DNA degradation in samples stored in ethanol was significantly influenced by heat treatment. This supports the idea that heat treating samples to temperatures around 80°C denatures DNA degrading enzymes. Regardless, DESS showed to be far more effective at inactivating such enzymes. Finally, the interaction between heat:temp for samples stored in ethanol further supports the idea that storage solution only affects DNA degradation when samples are heat treated. Again, this is likely caused by high initial levels of DNA degradation, masking the effect of storage temperature.
The better performance of DESS compared to ethanol can be observed in the fragment size distribution over time (Figure 1). DNA stored in ethanol was significantly more degraded after one day, while DNA stored in DESS appeared relatively stable over the first month. The drastic reduction in HWM DNA in ethanol after one day suggests enzymes were actively degrading the DNA. It is possible that the high concentration of ethanol has caused proteins close to the cell wall to coagulate too fast, creating a protein barrier that prevents the ethanol from reaching further into the cell. Consequently, enzymes continue to function, resulting in DNA degradation. Lower concentrations of ethanol (e.g. 70%) would allow the ethanol to reach further into the cell, however, this also reduces the effectiveness of the solution. Michaud and Foran (2011) tested three different concentrations of ethanol (i.e. 40, 70, and 100), but still found DESS to be most effective for preserving DNA. Further, the wide 95% confidence intervals do indicate a noticeable variation among individual samples. The observed variation is likely caused by the nested treatments within the experiment. Samples stored in DESS did show clear degradation after 3 months of storage. It is unclear what exact processes contributed to the observed degradation. It is possible that enzymes slowly start degrading the DNA over time, or chemical processes (e.g. hydrolyses) had become a contributing factor over time, or both.
The total amount of HMW DNA averaged per treatment group was < 16% (Table 3), raising the question as to what caused the initial stages of degradation. The physical handling of DNA during the extraction and pipetting processes can cause a mechanical break to double-stranded DNA. A protocol that limits the movement and manipulation of DNA would likely result in higher proportions or longer fragments (Mayjonade et al., 2016). This could be preferred a method when DNA is extracted for the sequencing of a reference genome, or long-read sequencing for detecting structural variation. A subset of the DNA would likely also have degraded within the first 24 hours after sampling. DNA was relatively stable for the first month when stored in DESS (Table 3), suggesting that degradation in the first 24 hours was limited. For the purposes of this study, the effects experienced in the initial 24 hours was not a specific interest, as the key goal was to evaluate how fast DNA degrades when molecular facilities are not at hand.
For this study we evaluated the performance of two commonly used preservation solutions, which offer an economical solution for sample preservation. Solutions such as RNAlater (Invitrogen©) and DNA/RNA shield (Zymo research©) provide additional options for sample preservation but come at an increased cost. These solutions are of increasing interest as they are capable of preserving both DNA and RNA. RNAlater works similar to DESS, where metal chelation by EDTA inactivates DNA degrading enzymes such as DNase. An important difference is that DMSO alters methylation profiles, rendering samples stored in DESS unfit for epigenetic research. The absence of DMSO in RNAlater and DNA/RNAshield potentially limits the transport of other components through cell membranes, which could be a problem for tough tissue samples. Another interesting feature of both RNAlater and DNA/RNA shield is that it has been designed to preserve DNA under ambient conditions. Also, lyophilisation (freeze drying) is a widely-applied method for the preservation of biological material. However, this method far less cost effective, and requires a specialist piece of equipment which is unpractical in the field. Finally, biostability molecules and dry-state DNA stabilization systems (e.g. Biomatrica®DNAstable® or polyvinyl alcohol, PVA) provide alternatives to the widely-applied TE buffer for long-term storage of purified DNA (Clement et al., 2012, Ivanova & Kuzmina, 2013). These compounds have been found to preserve purified DNA better at non-freezing temperatures, which can be particularly useful when shipping DNA over long-distances to sequencing facilities.
Conclusions
The application of NGS will continue to increase over the coming decade, and an increasing number of studies will be conducted on non-model species sampled in the field. Ongoing reductions in sequencing costs and the large selection of services offered by sequencing providers (from DNA extraction to genome annotation) are making genomic research accessible to a large scientific audience. Sample collection and preservation is the first and crucial step that will allow us to gain novel insights regarding animal ecology, demography, and evolution using genomic methods. Our study highlights the need for careful evaluation of sample preservation and provides key considerations for anyone planning sampling DNA for genomic research.
Acknowledgements
This work was supported by New Zealand Marsden Fund project 16‐VUW‐040. We would like to thank The New Zealand Institute for Plant and Food Research Ltd staff who helped with collecting the tissue samples, and Bastiaan Star for feedback on the manuscript. We also thank Monica Holland, Chris Kirk and Igor Ruza for providing useful corrections and comments on the manuscript.
Data accessibility
All data files, python and R scripts are available in the supplementary information.
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