The effect of DNA extraction methodology on gut microbiota research applications.
The effect of DNA extraction methodology on gut microbiota research applications.
复制标题
DOI：
10.1186/s13104-016-2171-7
复制
发表时间：
2016-07-26
影响因子：
1.8
通讯作者：
Ijaz UZ
中科院分区：
文献类型：
--
作者：
Gerasimidis K;Bertz M;Quince C;Brunner K;Bruce A;Combet E;Calus S;Loman N;Ijaz UZ
传统和现代 DNA 提取方法对研究肠道微生物群在健康和疾病中的作用的应用的影响是当前感兴趣的话题。使用三种流行的方法从三份粪便样本和一份益生菌胶囊中提取基因组 DNA；离液 (CHAO) 方法、苯酚/氯仿 (PHEC) 萃取、专有试剂盒 (QIAG)。评估了每种方法在 DNA 产量和质量、使用定量 PCR 的微生物群组成、16S rRNA 基因深度测序以及测序分析流程方面的性能。 CHAO 方法的双链 DNA 产量最高，QIAG 试剂盒的双链 DNA 量最低，但后一种方法分离核酸的纯度更好。 CHAO 方法产生每质量 (g) 粪便的细菌类群浓度较高。与其他方法相比，CHAO 方法的测序覆盖率较高，但保留了较高比例的初始测序读数以分配给 QIAG 试剂盒中的操作分类单位 (OTU)。与其他两种方法相比，QIAG 试剂盒似乎具有较长的修剪读数和较短的质量较差的区域。未观察到不同 DNA 提取方法之间 α 多样性指数的明显分离。当探索样品之间的成分差异时，根据样品类型观察到强烈的分离。提取方法的效果要么很小（Bray-Curtis 距离），要么没有（未加权 Unifrac 距离）。每个样本中的分类单元成员资格和丰度与所使用的 DNA 提取方法无关。我们对肠道微生物群研究中常用的几种 DNA 提取方法进行了基准测试，它们的差异取决于预期使用的下游应用。当打算汇集和分析使用不同 DNA 提取方法的研究中的样本或数据时，应小心谨慎。本文的在线版本 (doi:10.1186/s13104-016-2171-7) 包含补充材料，可供授权用户使用。
The effect that traditional and modern DNA extraction methods have on applications to study the role of gut microbiota in health and disease is a topic of current interest. Genomic DNA was extracted from three faecal samples and one probiotic capsule using three popular methods; chaotropic (CHAO) method, phenol/chloroform (PHEC) extraction, proprietary kit (QIAG). The performance of each of these methods on DNA yield and quality, microbiota composition using quantitative PCR, deep sequencing of the 16S rRNA gene, and sequencing analysis pipeline was evaluated. The CHAO yielded the highest and the QIAG kit the lowest amount of double-stranded DNA, but the purity of isolated nucleic acids was better for the latter method. The CHAO method yielded a higher concentration of bacterial taxa per mass (g) of faeces. Sequencing coverage was higher in CHAO method but a higher proportion of the initial sequencing reads were retained for assignments to operational taxonomic unit (OTU) in the QIAG kit compared to the other methods. The QIAG kit appeared to have longer trimmed reads and shorter regions of worse quality than the other two methods. A distinct separation of α-diversity indices between different DNA extraction methods was not observed. When compositional dissimilarities between samples were explored, a strong separation was observed according to sample type. The effect of the extraction method was either marginal (Bray–Curtis distance) or none (unweighted Unifrac distance). Taxon membership and abundance in each sample was independent of the DNA extraction method used. We have benchmarked several DNA extraction methods commonly used in gut microbiota research and their differences depended on the downstream applications intended for use. Caution should be paid when the intention is to pool and analyse samples or data from studies which have used different DNA extraction methods. The online version of this article (doi:10.1186/s13104-016-2171-7) contains supplementary material, which is available to authorized users.