Comparing sediment DNA extraction methods for assessing organic enrichment associated with marine aquaculture.
Comparing sediment DNA extraction methods for assessing organic enrichment associated with marine aquaculture.
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作者：
Pearman JK;Keeley NB;Wood SA;Laroche O;Zaiko A;Thomson-Laing G;Biessy L;Atalah J;Pochon X
海洋沉积物中含有丰富多样的微生物和大型生物，它们对生物地球化学循环的功能起着重要作用。传统上，人为扰动是通过识别沿梯度的宏观生物响应来研究的。环境DNA （Environmental DNA, eDNA）分析最近被提倡作为一种快速和经济有效的方法来测量生态影响，并且正在努力将eDNA工具纳入监测中。在这些方法取代或补充现有方法之前，必须证明每个分析步骤的稳健性和可重复性。需要进一步研究的一个领域是沉积物DNA提取方法的选择。在新西兰Otanerau湾，沿一个奇努克鲑鱼养殖场附近的扰动梯度采集了环境DNA沉积物样本。采用四种提取试剂盒（Qiagen DNeasy PowerSoil、Qiagen DNeasy PowerSoil Pro、Qiagen RNeasy PowerSoil总RNA/DNA提取/洗脱试剂盒和Favorgen FavorPrep Soil DNA分离Midi Kit）和三种沉淀物体积（0.25、2和5 g）提取DNA。利用引物分别针对16S和18S核糖体RNA基因扩增原核和真核生物群落，并在Illumina MiSeq上测序。从每个提取试剂盒中获得多样性和群落组成估计值，以及它们在已建立的元条形码生物指数中的相对表现。两种试剂盒提取的DNA在质量和数量上存在差异，其中Qiagen dnneasy PowerSoil试剂盒的提取效果最好。不同试剂盒间原核生物和真核生物丰富度差异显著（p < 0.001）。一小部分扩增子序列变异（asv）在试剂盒中是共享的（约3%），尽管这些共享的asv占大部分序列读长（原核生物：59.9%，真核生物：67.2%）。每个试剂盒显示的分类类的丰富度和相对丰度存在差异。多变量分析表明，“离农场距离”和“工具包”在解释群落组成方面存在显著的交互作用，其中离农场距离是群落组成的一个更强的决定因素。与细菌和真核生物元条形码生物指数的比较表明，所有试剂盒在环境梯度上表现出相似的模式。总的来说，我们提倡使用Qiagen dnasy PowerSoil试剂盒来表征海洋农场沉积物中的原核和真核eDNA。我们的结论是基于与本研究中调查的其他试剂盒/数量相比，这些试剂盒获得的DNA质量值和丰富度更高。PowerSoil试剂盒的另一个优点是DNA提取可以使用提取器机器人进行，提供了额外的标准化和结果的可重复性。
Marine sediments contain a high diversity of micro- and macro-organisms which are important in the functioning of biogeochemical cycles. Traditionally, anthropogenic perturbation has been investigated by identifying macro-organism responses along gradients. Environmental DNA (eDNA) analyses have recently been advocated as a rapid and cost-effective approach to measuring ecological impacts and efforts are underway to incorporate eDNA tools into monitoring. Before these methods can replace or complement existing methods, robustness and repeatability of each analytical step has to be demonstrated. One area that requires further investigation is the selection of sediment DNA extraction method. Environmental DNA sediment samples were obtained along a disturbance gradient adjacent to a Chinook (Oncorhynchus tshawytscha) salmon farm in Otanerau Bay, New Zealand. DNA was extracted using four extraction kits (Qiagen DNeasy PowerSoil, Qiagen DNeasy PowerSoil Pro, Qiagen RNeasy PowerSoil Total RNA/DNA extraction/elution and Favorgen FavorPrep Soil DNA Isolation Midi Kit) and three sediment volumes (0.25, 2, and 5 g). Prokaryotic and eukaryotic communities were amplified using primers targeting the 16S and 18S ribosomal RNA genes, respectively, and were sequenced on an Illumina MiSeq. Diversity and community composition estimates were obtained from each extraction kit, as well as their relative performance in established metabarcoding biotic indices. Differences were observed in the quality and quantity of the extracted DNA amongst kits with the two Qiagen DNeasy PowerSoil kits performing best. Significant differences were observed in both prokaryotes and eukaryotes (p < 0.001) richness among kits. A small proportion of amplicon sequence variants (ASVs) were shared amongst the kits (~3%) although these shared ASVs accounted for the majority of sequence reads (prokaryotes: 59.9%, eukaryotes: 67.2%). Differences were observed in the richness and relative abundance of taxonomic classes revealed with each kit. Multivariate analysis showed that there was a significant interaction between “distance” from the farm and “kit” in explaining the composition of the communities, with the distance from the farm being a stronger determinant of community composition. Comparison of the kits against the bacterial and eukaryotic metabarcoding biotic index suggested that all kits showed similar patterns along the environmental gradient. Overall, we advocate for the use of Qiagen DNeasy PowerSoil kits for use when characterizing prokaryotic and eukaryotic eDNA from marine farm sediments. We base this conclusion on the higher DNA quality values and richness achieved with these kits compared to the other kits/amounts investigated in this study. The additional advantage of the PowerSoil Kits is that DNA extractions can be performed using an extractor robot, offering additional standardization and reproducibility of results.
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影响因子：
9.7
作者：
Holmsgaard, Peter N.;Norman, Anders;Sorensen, Soren J.
通讯作者：
Sorensen, Soren J.
影响因子：
48
作者：
Callahan BJ;McMurdie PJ;Rosen MJ;Han AW;Johnson AJ;Holmes SP
通讯作者：
Holmes SP
影响因子：
4.4
作者：
HOLBEN, WE;JANSSON, JK;TIEDJE, JM
通讯作者：
TIEDJE, JM
影响因子：
4.5
作者：
Dell'Anno, A;Bompadre, S;Danovaro, R
通讯作者：
Danovaro, R
影响因子：
5
作者：
Carrigg, Cora;Rice, Olivia;O'Flaherty, Vincent
通讯作者：
O'Flaherty, Vincent