Utilizing field collected insects for next generation sequencing: Effects of sampling, storage, and DNA extraction methods

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.

A DNA barcode-based survey of wild urban bees in the Loire Valley, France

“…However, the presence of numts could contribute to failures in sequencing or the sequencing of contaminants. Sampling, storage conditions and DNA extraction methods can be at the origin of a number of sequencing problems 64 . Our pan-trapping involved bees being kept in soapy water for up to 4 days.…”

Genome wide identification of QTL associated with yield and yield components in two popular wheat cultivars TAM 111 and TAM 112

“…SNP calling was processed as described by Yang et al [ 22 ]. More details are described below with some modification from Elshire et al [ 23 ], Poland et al [ 24 ], and Ballare et al [ 25 ].…”

Efficient assessment of nocturnal flying insect communities by combining automatic light traps and DNA metabarcoding

“…Besides moths, the trap was also able to capture a wide diversity of other arthropods, in particular Diptera, Coleoptera, Hemiptera, and even Orthoptera, proving its suitability to study insects capable of nocturnal flight, this way complementing Malaise trap‐based studies. Furthermore, our trap was adapted to retain insects without the need to use any chemical product to kill the individuals, which likely facilitates the subsequent steps of DNA extraction and amplification (Ballare et al., 2019; Dillon et al., 1996). Finally, the use of a breathable fabric bag makes it easier to extract insects from the trap and to undertake the initial processing steps in the field with minimal handling of samples, thereby reducing the risks of contamination across samples.…”

“…However, efforts are still needed to develop and optimize traps that can be efficiently combined with metabarcoding in large scale field surveys. Methods that can avoid the need of hand‐picking individual specimens, thus reducing effort and contamination risks, and the use of chemical compounds to kill or otherwise retain insects within traps, which would make the subsequent steps of DNA extraction and amplification more difficult (Ballare et al., 2019; Dillon, Austin, & Bartowsky, 1996), would be particularly useful.…”