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

2019Artigo científico Acesso aberto
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dc.contributor.authorBallare, K.M.
dc.contributor.authorPope, N.S.
dc.contributor.authorCastilla, A.R.
dc.contributor.authorCusser, S.
dc.contributor.authorMetz, R.P.
dc.contributor.authorJha, S.
dc.date.accessioned2020-01-24T12:23:09Z
dc.date.available2020-01-24T12:23:09Z
dc.date.issued2019
dc.description.abstractDNA 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 analysespt_PT
dc.description.versioninfo:eu-repo/semantics/publishedVersionpt_PT
dc.identifier.citationEcology and Evolution. 2019;9:13690–13705pt_PT
dc.identifier.doiDOI: 10.1002/ece3.5756pt_PT
dc.identifier.urihttp://hdl.handle.net/10400.5/19420
dc.language.isoengpt_PT
dc.publisherWileypt_PT
dc.relationDEB‐0908661pt_PT
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/pt_PT
dc.subjectcurated insectspt_PT
dc.subjectddRADpt_PT
dc.subjectdegraded DNApt_PT
dc.subjectnext generation sequencingpt_PT
dc.subjectpan trapspt_PT
dc.subjectpropylene glycolpt_PT
dc.titleUtilizing field collected insects for next generation sequencing: effects of sampling, storage and DNA extraction methodspt_PT
dc.typejournal article
dspace.entity.typePublication
oaire.citation.titleEcology and Evolutionpt_PT
rcaap.rightsopenAccesspt_PT
rcaap.typearticlept_PT

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