Massachusetts Institute of Technology-Woods Hole Oceanographic Institution Joint Program in Oceanography/Applied Ocean Science and Engineering, Cambridge, MA, 02139, USA.
Woods Hole Oceanographic Institution, Marine Chemistry and Geochemistry, Woods Hole, MA, 02543, USA.
Microbiome. 2017 Feb 8;5(1):18. doi: 10.1186/s40168-017-0229-y.
DNA-based sequencing approaches are commonly used to identify microorganisms and their genes and document trends in microbial community diversity in environmental samples. However, extraction of microbial DNA from complex environmental samples like corals can be technically challenging, and extraction methods may impart biases on microbial community structure.
We designed a two-phase study in order to propose a comprehensive and efficient method for DNA extraction from microbial cells present in corals and investigate if extraction method influences microbial community composition. During phase I, total DNA was extracted from seven coral species in a replicated experimental design using four different MO BIO Laboratories, Inc., DNA Isolation kits: PowerSoil®, PowerPlant® Pro, PowerBiofilm®, and UltraClean® Tissue & Cells (with three homogenization permutations). Technical performance of the treatments was evaluated using DNA yield and amplification efficiency of small subunit ribosomal RNA (SSU ribosomal RNA (rRNA)) genes. During phase II, potential extraction biases were examined via microbial community analysis of SSU rRNA gene sequences amplified from the most successful DNA extraction treatments.
In phase I of the study, the PowerSoil® and PowerPlant® Pro extracts contained low DNA concentrations, amplified poorly, and were not investigated further. Extracts from PowerBiofilm® and UltraClean® Tissue and Cells permutations were further investigated in phase II, and analysis of sequences demonstrated that overall microbial community composition was dictated by coral species and not extraction treatment. Finer pairwise comparisons of sequences obtained from Orbicella faveolata, Orbicella annularis, and Acropora humilis corals revealed subtle differences in community composition between the treatments; PowerBiofilm®-associated sequences generally had higher microbial richness and the highest coverage of dominant microbial groups in comparison to the UltraClean® Tissue and Cells treatments, a result likely arising from using a combination of different beads during homogenization.
Both the PowerBiofilm® and UltraClean® Tissue and Cells treatments are appropriate for large-scale analyses of coral microbiota. However, studies interested in detecting cryptic microbial members may benefit from using the PowerBiofilm® DNA treatment because of the likely enhanced lysis efficiency of microbial cells attributed to using a variety of beads during homogenization. Consideration of the methodology involved with microbial DNA extraction is particularly important for studies investigating complex host-associated microbiota.
基于 DNA 的测序方法常用于鉴定微生物及其基因，并记录环境样本中微生物群落多样性的趋势。然而，从珊瑚等复杂环境样本中提取微生物 DNA 在技术上具有挑战性，并且提取方法可能会对微生物群落结构产生偏差。
我们设计了一项两阶段研究，旨在提出一种从珊瑚中存在的微生物细胞中提取 DNA 的综合高效方法，并研究提取方法是否会影响微生物群落组成。在第一阶段，我们使用四种不同的 MO BIO 实验室公司 DNA 分离试剂盒（PowerSoil®、PowerPlant® Pro、PowerBiofilm® 和 UltraClean® Tissue & Cells（有三种均化处理），在重复实验设计中从七种珊瑚物种中提取总 DNA。通过小亚基核糖体 RNA（SSU 核糖体 RNA（rRNA））基因的 DNA 产量和扩增效率评估处理的技术性能。在第二阶段，通过对从最成功的 DNA 提取处理中扩增的 SSU rRNA 基因序列进行微生物群落分析，检查潜在的提取偏差。
在研究的第一阶段，PowerSoil®和 PowerPlant® Pro 提取物的 DNA 浓度低、扩增效果差，因此没有进一步研究。在第二阶段进一步研究了 PowerBiofilm®和 UltraClean® Tissue 和 Cells 排列的提取物，序列分析表明，微生物群落组成总体上取决于珊瑚物种，而不是提取处理。对来自 Orbicella faveolata、Orbicella annularis 和 Acropora humilis 珊瑚的序列进行更精细的两两比较表明，处理之间的群落组成存在细微差异；与 UltraClean® Tissue 和 Cells 处理相比，PowerBiofilm®相关序列通常具有更高的微生物丰富度和优势微生物组的最高覆盖率，这一结果可能是由于在均化过程中使用了不同的珠子组合。
PowerBiofilm®和 UltraClean® Tissue 和 Cells 两种处理方法均适用于大规模珊瑚微生物组分析。然而，对于检测隐式微生物成员的研究，使用 PowerBiofilm® DNA 处理可能会受益，因为在均化过程中使用多种珠子可能会提高微生物细胞的裂解效率。考虑微生物 DNA 提取所涉及的方法对于研究复杂的宿主相关微生物群尤为重要。
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