DNA preservation &amp; DNA extraction protocol for field collection of coral samples suitable for host-, marker gene-, and metagenomics-based sequencing approaches
Creators: name: Voolstra, Christian R
name: Colin, Luigi
name: Dörr, Melanie S
name: Cárdenas, Anny
name: Perna, Gabriela
name: Fiesinger, Anna
Publisher: name: Zenodo
Year: 2023
Subjects: coral
DNA isolation
DNA storage
field work
sample preservation

description: <strong>Summary</strong> The ability to collect samples suitable for DNA extraction in remote field settings without the necessity of freezing is a common need for molecular ecologists that has been addressed by the so-called DESS buffer, a solution containing DMSO, EDTA, and saturated NaCl (Dawson, Raskoff, &amp; Jacobs, 1998; Seutin, White, &amp; Boag, 1991). DESS is commonly used as a storage buffer for coral samples (Pinzón et al., 2013; Voolstra et al., 2021; Ziegler et al., 2017) and has also been shown to faithfully preserve DNA for bacterial community analyses (Gray, Pratte, &amp; Kellogg, 2013; Lee, Adams, &amp; Klassen, 2019). What is lesser known is that DESS buffer has been shown to preserve nematode morphology, comparable to formalin-based fixatives (Yoder et al., 2006) and, as such, preserves cell integrity. This makes it suitable for metagenomics applications, which typically rely on differentially lysis of eukaryotic (host) and prokaryotic (microbiome) cells to effectively deplete the eukaryotic host DNA. Protocols for effective depletion of coral host DNA and enrichment of bacterial DNA coverage for application in metagenome shotgun sequencing studies to our knowledge do not exist (Neave, Michell, Apprill, &amp; Voolstra, 2017; Robbins et al., 2019). To address this, we here developed a protocol for coral DNA preservation and DNA extraction suitable for host- (e.g., sWGS), marker gene- (e.g., ITS2, 16S), and metagenomics-based (e.g., microbiome) sequencing approaches. The protocol employs the DNA- and cell morphology-/integrity-preserving properties of DESS buffer, compatible with application in remote field settings and evading the need for cooling/freezing of samples. Sprayed-off tissue from coral fragments is then used for DNA extraction using two distinct extraction kits (the Qiagen DNeasy Blood &amp; Tissue Kit and the Qiagen QIAamp DNA Microbiome Kit) for use in host-, marker gene-, and metagenomics-based sequencing approaches. The protocol was successfully applied to coral samples from <em>Acropora</em> sp., <em>Pocillopora</em> sp., and <em>Porites</em> sp.. A detailed study on performance, efficacy, and benchmarking of the here-developed protocol in comparison to a range of other protocols is in preparation and is anticipated to be available shortly. We hope the here-developed protocol applies broadly and will be used widely.
description: Changes to Version 1.0: option to spray off tissue in initial DESS buffer volume was added for small sample volumes ≤ 2 mL. --- Changes to Version 1.1: added coral tissue homogenization step in DESS buffer before proceeding with either DNA isolation protocol --- Changes to Version 1.2: added a bead-beating step to cracking/opening Symbiodiniaceae cells for efficient algal DNA lysis; added proteinase K digestion step following Benzonase-based extracellular eukaryotic DNA lysis. NOTE: the DOCX file says Version 1.2 instead of Version 1.3. The mistake will be rectified in a future release. --- This research was supported by funding from the University of Konstanz (AFF) and the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) project number 458901010.
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