Abstract:
The more demanding requirements of DNA preservation for genomic research
can be difficult to meet when field conditions limit the methodological
approaches that can be used, or cause samples to be stored in suboptimal
conditions. Such limitations may increase rates of DNA degradation,
potentially rendering samples unusable for applications such as
genome-wide sequencing. Nonetheless, little is known about the impact of
suboptimal sampling conditions. We evaluated the performance of two
widely used preservation solutions (1. DESS: 20% DMSO, 0.25M EDTA, NaCl
saturated solution, and 2. ethanol) under a range of storage conditions
over a three-month period (sampling at 1 day, 1 week, 2 weeks, 1 month,
and 3 months) to provide practical guidelines for DNA preservation. DNA
degradation was quantified as the reduction in average DNA fragment size
over time (DNA fragmentation) because the size distribution of DNA
segments plays a key role in generating genomic datasets. Tissues were
collected from a marine teleost species, the Australasian snapper,Chrysophrys auratus. We found that the storage solution has a
dramatic effect on DNA preservation. In DESS, DNA was only moderately
degraded after three months of storage while DNA stored in ethanol
showed high levels of DNA degradation already within 24 hours, making
samples unsuitable for next-generation-sequencing. We recommend DESS as
the most promising solution to improve DNA preservation. These results
provide practical and economical advice to improve DNA preservation when
sampling for genome-wide applications.
Keywords: DMSO, DNA preservation, ethanol, fish,
next-generation-sequencing, NGS, snapper
Introduction
Next-generation sequencing (NGS) applications are now widely applied to
population genomic studies of non-model species, addressing questions
related to the conservation, ecology, evolution, and demography of
species (Ellegren, 2014). These applications typically require
high-molecular-weight (HMW) DNA (>20 kbp) for library
preparation and sequencing to obtain high-quality genomic datasets. Such
requirements are more demanding than those for traditional PCR-based
approaches where a wide range of DNA qualities can be used, as targeted
DNA sequences are seldom longer than 5 kbp. As a consequence, earlier
methods for preserving samples for genetic analysis may perform
suboptimally and fail to meet requirements of DNA preservation for
genomic research.
DNA is ideally extracted immediately after tissue sampling, or stored at
sub-zero temperatures and extracted shortly after (e.g. within 24
hours). However, field conditions and limited funding often restrict the
resources available for sample preservation. This forces researchers to
work within tight boundaries to prevent DNA degradation as much as
possible. The level of preservation required to obtain high-quality DNA
will vary depending on the goals of the study as well as environmental
conditions, time spent in the field, available resources, and type of
tissue collected. The solutions in which tissues are preserved provide
another layer of added flexibility. Two of the most common and also
cost-effective solutions to preserve tissue for DNA extraction are
ethanol and DESS (20% DMSO, 0.25M EDTA, NaCl saturated solution) (Yoder
et al., 2006). However, it is less clear how suitable these solutions
are for genomic research when preservation conditions are variable and
when the period over which tissues are stored spans multiple months.
Previous studies that assessed sample preservation using various storage
solutions had quantified DNA quality based on the quantity extracted, or
ability to sequence short DNA fragments (<5 kbp) (Asahida et
al., 1996, Bainard et al., 2010, Dawson et al., 1998, Gorokhova, 2005,
Graham et al., 2015, Graham et al., 2008, Michaud & Foran, 2011, Seutin
et al., 1991, Stein et al., 2013). Such metrics do not provide an
accurate assessment of DNA quality for most genomic sequencing
applications, as they do not provide any information regarding integrity
of the DNA on large scales. Among the studies mentioned above, time
periods over which sample preservation was assessed varied greatly,
spanning from 12 hours up to three years (Graham et al., 2015, Graham et
al., 2008). When included in the study design, DESS solution was found
to preserve DNA best according to the quality metrics of the study in
question (Dawson et al., 1998, Michaud & Foran, 2011, Seutin et al.,
1991). We chose DNA fragment size as a proxy for DNA quality because it
is a key metric used by sequencing providers for generating
representative genomic datasets with practically any sequencing
platform. Here, we will refer to DNA degradation as double-stranded
breaks resulting in a reduction in average fragment size. DNA changes
that do not result in a reduction in fragment size will be referred to
as DNA damage.