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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