Ezzy Alan C, Hagstrom Amanda D, George Chris, Hamlin Adam S, Pereg Lily, Murphy Aron J, Winter Gal
School of Science and Technology, the University of New England, Armidale, NSW 2351, Australia.
School of Science and Technology, the University of New England, Armidale, NSW 2351, Australia; School of Medical Sciences, The University of New South Wales, Sydney 2052, Australia.
J Microbiol Methods. 2019 Sep;164:105668. doi: 10.1016/j.mimet.2019.105668. Epub 2019 Jul 11.
Human gut microbiome analysis through faecal sampling typically involves five stages: sample collection, storage, DNA extraction, next generation sequencing and bioinformatics analysis. Of these, the first three are considered irreversible. This feasibility study describes an assessment of methodologies used for faecal DNA extraction and sample handling, using the parameters DNA yield, purity and resultant microbial profile. Six DNA extraction techniques, including commercially available kits and manual protocols were compared on human faecal samples (n = 3). Different extraction techniques produced significant variance in DNA yield (range 2.7-164 ng/mg faeces) and microbial diversity profiles, with considerable variation in phyla dominance (Firmicutes (P < 0.001), Bacteroidetes (P = 0.003), Actinobacteria (P = 0.003), One-way ANOVA). The most effective method, with the highest DNA yield, was a simple and inexpensive extraction technique named MetaHIT. Using this method, DNA was extracted from separate faecal samples (n = 3) and had been aliquoted to seven storage conditions including three stabilizing buffers and three temperature conditions, for a period of 120-h, with storage at -80 °C as a control treatment. DNA yield and purity was not statistically different between the control and remaining treatments. 16S rDNA-based diversity profile was largely comparable across the treatments with only minor differences in genera between samples stored at room temperature in air and - 80 °C control. Overall these results suggest that the choice of DNA extraction method has a greater influence on the resultant microbial diversity profile than the short-term storage method.
样本采集、储存、DNA提取、下一代测序和生物信息学分析。其中，前三个阶段被认为是不可逆的。本可行性研究描述了使用DNA产量、纯度和所得微生物谱参数对粪便DNA提取和样本处理方法的评估。对六种DNA提取技术进行了比较，包括市售试剂盒和手动方法，分析对象为人类粪便样本（n = 3）。不同的提取技术在DNA产量（范围为2.7 - 164 ng/mg粪便）和微生物多样性谱方面产生了显著差异，门水平的优势度存在相当大的变化（厚壁菌门（P < 0.001）、拟杆菌门（P = 0.003）、放线菌门（P = 0.003），单因素方差分析）。最有效的方法，即DNA产量最高的方法，是一种名为MetaHIT的简单且廉价的提取技术。使用该方法从单独的粪便样本（n = 3）中提取DNA，并将其分装到七种储存条件下，包括三种稳定缓冲液和三种温度条件，持续120小时，以-80°C储存作为对照处理。对照处理与其余处理之间的DNA产量和纯度在统计学上没有差异。基于16S rDNA的多样性谱在各处理之间基本可比，仅在室温空气中储存的样本与-80°C对照样本之间的属水平存在微小差异。总体而言，这些结果表明，DNA提取方法的选择对所得微生物多样性谱的影响大于短期储存方法。
