DOI: 10.7291/d1cd4p
title: Utilizing field collected insects for next generation sequencing: effects of sampling, storage, and DNA extraction methods

description: 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 (&gt;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.
description: Specimens were
 captured using three standardized methods including hand netting, blue
 vane trapping (Stephen & Rao 2007), and pan trapping
 (Roulston <i>et al.</i> 2007) at 39 sites across Texas during the
 summers of 2012-2014. All specimens were extracted in Spring 2016 using
 Qiagen ® DNeasy Blood and Tissue Kit using the standard protocol with a
 few minor modifications to maximize DNA yield. We extracted approximately
 1 cm<sup>3</sup> tissue from each specimen, using thoracic
 tissue from <i>B. pensylvanicus </i>and<i> M.
 tepaneca</i>, and using the entire specimen for the smaller species
 <i>L. bardum</i>. . We additionally investigated the DNAzol ®
 extraction technique for only <i>B. pensylvanicus</i>
 specimens given the larger size and ample tissue availability. <i>B.
 pensylvanicus</i> thoraces were divided in half and extracted using
 a customized DNAzol ® protocol (Chomczynski <i>et
 al.</i> 1997). One
 hundred ng of DNA per sample after normalization using PicoGreen ®
 measurement were digested in 1X NEB Cut Smart Buffer and 100 U each
 EcoRI-HF and MspI (NEB), for a final volume of 25 µL, at 37°C for 4
 hours.  Sequencing of all 136 samples was done across two lanes of
 Illumina HiSeq 2500 operated by the Genomics and Bioinformatics service at
 Texas A&M University (TAMU), with 125 million reads per lane.
 De-multiplexing of sequencing reads, trimming of adapters, and removal of
 barcodes, was performed using bclfastq2 v2.19 (Illumina).
description: Data includes: 1) a CSV file with
 individual sample names, sequence read names, treatments, and DNA quality
 metrics (NanoDrop indices and DNA concentration); 2) tar archive of R data
 analysis scripts; 3) and tar archives of assembly files and STACKS outputs
 for <em>Bombus pensylvanicus</em>, <em>Melissodes
 tepaneca</em>, and <em>Lasioglossum bardum</em> ddRAD
 assemblies respectively.  Each tar archive contains ReadMe files which
 describe the individual files and usage. See Methods section in Ballare
 <em>et al. </em>(2019) <em>Ecology and
 Evolution</em> for further details on analysis methods.