Patin Nastassia Virginia, Goodwin Kelly D
Atlantic Oceanographic and Meteorological Laboratory, Ocean Chemistry and Ecosystems Division, National Oceanic and Atmospheric Administration, Miami, FL, United States.
Cooperative Institute for Marine and Atmospheric Studies, Rosenstiel School of Marine, Atmospheric, and Earth Science, University of Miami, Miami, FL, United States.
Front Microbiol. 2023 Jan 12;13:1026596. doi: 10.3389/fmicb.2022.1026596. eCollection 2022.
The expanding interest in marine microbiome and eDNA sequence data has led to a demand for sample collection and preservation standard practices to enable comparative assessments of results across studies and facilitate meta-analyses. We support this effort by providing guidelines based on a review of published methods and field sampling experiences. The major components considered here are environmental and resource considerations, sample processing strategies, sample storage options, and eDNA extraction protocols. It is impossible to provide universal recommendations considering the wide range of eDNA applications; rather, we provide information to design fit-for-purpose protocols. To manage scope, the focus here is on sampling collection and preservation of prokaryotic and microeukaryotic eDNA. Even with a focused view, the practical utility of any approach depends on multiple factors, including habitat type, available resources, and experimental goals. We broadly recommend enacting rigorous decontamination protocols, pilot studies to guide the filtration volume needed to characterize the target(s) of interest and minimize PCR inhibitor collection, and prioritizing sample freezing over (only) the addition of preservation buffer. An annotated list of studies that test these parameters is included for more detailed investigation on specific steps. To illustrate an approach that demonstrates fit-for-purpose methodologies, we provide a protocol for eDNA sampling aboard an oceanographic vessel. These guidelines can aid the decision-making process for scientists interested in sampling and sequencing marine microbiomes and/or eDNA.
对海洋微生物组和环境DNA序列数据的兴趣不断增加,这导致了对样本采集和保存标准方法的需求,以便能够对各项研究的结果进行比较评估,并促进荟萃分析。我们通过回顾已发表的方法和实地采样经验,提供指导方针来支持这项工作。这里考虑的主要内容包括环境和资源因素、样本处理策略、样本储存选项以及环境DNA提取方案。鉴于环境DNA应用的广泛范围,不可能提供通用建议;相反,我们提供信息以设计适用的方案。为了控制范围,这里的重点是原核和微型真核生物环境DNA的采样收集和保存。即使是在有限的视角下,任何方法的实际效用也取决于多种因素,包括栖息地类型、可用资源和实验目标。我们广泛建议制定严格的去污方案,开展试点研究以指导表征目标所需的过滤体积,并尽量减少PCR抑制剂的收集,优先考虑样本冷冻而非仅添加保存缓冲液。还包含一份测试这些参数的研究注释列表,以便对具体步骤进行更详细的研究。为了说明一种展示适用方法的途径,我们提供了一份在海洋学船上进行环境DNA采样的方案。这些指导方针有助于对海洋微生物组和/或环境DNA进行采样和测序的科学家进行决策。