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.