Abstract
DNA sequencing technologies continue to advance the biological sciences, expanding opportunities for genomic studies of non‐model organisms for basic and applied questions. Despite these opportunities, many next generation sequencing protocols have been developed assuming a substantial quantity of high molecular weight DNA (>100 ng), which can be difficult to obtain for many study systems. In particular, the ability to sequence field‐collected specimens that exhibit varying levels of DNA degradation remains largely unexplored. In this study we investigate the influence of five traditional insect capture and curation methods on Double‐Digest Restriction Enzyme Associated DNA (ddRAD) sequencing success for three wild bee species. We sequenced a total of 105 specimens (between 7–13 specimens per species and treatment). We additionally investigated how different DNA quality metrics (including pre‐sequence concentration and contamination) predicted downstream sequencing success, and also compared two DNA extraction methods. We report successful library preparation for all specimens, with all treatments and extraction methods producing enough highly reliable loci for population genetic analyses. Although results varied between species, we found that specimens collected by net sampling directly into 100% EtOH, or by passive trapping followed by 100% EtOH storage before pinning tended to produce higher quality ddRAD assemblies, likely as a result of rapid specimen desiccation. Surprisingly, we found that specimens preserved in propylene glycol during field sampling exhibited lower‐quality assemblies. We provide recommendations for each treatment, extraction method, and DNA quality assessment, and further encourage researchers to consider utilizing a wider variety of specimens for genomic analyses.
Highlights
The rapid expansion of molecular techniques throughout the last 60 years has revolutionized the field of biology and the study of wild populations across taxonomic and spatial scales
Other studies have shown that degraded DNA from museum specimens can be successfully used for RAD procedures (Haponski, Lee, & Foighil,2017; Sproul & Maddison, 2017; Tin, Economo, & Mikheyev, 2014), but these studies have not examined the impacts of differing sampling techniques, different DNA extraction methods, or use of different focal species on Double‐Digest Restriction Enzyme Associated DNA (ddRAD) sequencing success
In this study we investigate the effects of capture method, preservation method, and DNA extraction method on ddRAD se‐ quencing success for three wild bee species: Bombus pensylvanicus, Melissodes tepaneca, and Lasioglossum bardum
Summary
The rapid expansion of molecular techniques throughout the last 60 years has revolutionized the field of biology and the study of wild populations across taxonomic and spatial scales. In a recent review of NGS methods, approximately 80% of the studies cited used tissue that was either freshly sampled or preserved in EtOH (Andrews, Good, Miller, Luikart, & Hohenlohe, 2016) This pattern belies the difficulty of obtaining high quality DNA for many species, the greater availability of specimens that may have lower quality DNA, and the untapped potential of these sam‐ ples to address key questions in ecology and evolution. Other studies have shown that degraded DNA from museum specimens (collected 50–100 years ago) can be successfully used for RAD procedures (Haponski, Lee, & Foighil,2017; Sproul & Maddison, 2017; Tin, Economo, & Mikheyev, 2014), but these studies have not examined the impacts of differing sampling techniques (e.g. netting vs trapping), different DNA extraction methods, or use of different focal species on ddRAD sequencing success. We expected that trapped specimens which included propylene glycol as a preser‐ vative would have higher DNA quality and better sequencing results than those specimens trapped without a preservative
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