Back to Basics--The Influence of DNA Extraction and Primer Choice on Phylogenetic Analysis of Activated Sludge Communities.
Back to Basics--The Influence of DNA Extraction and Primer Choice on Phylogenetic Analysis of Activated Sludge Communities.
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DOI：
10.1371/journal.pone.0132783
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Nielsen PH
中科院分区：
文献类型：
--
作者：
Albertsen M;Karst SM;Ziegler AS;Kirkegaard RH;Nielsen PH
在所有微生物扩增子测序分析中，DNA提取和引物选择对观察到的群落结构有很大影响。虽然偏见是众所周知的，没有全面的分析已经在活性污泥社区进行。在这项研究中，我们系统地探讨了一些参数对所观察到的微生物群落的影响：珠击强度，引物选择，细胞外DNA去除，和各种PCR设置。总共对176个样本进行了16 S rRNA扩增子测序，并通过宏基因组学和元转录组学对选定的样本进行了研究。定量荧光原位杂交作为一种DNA提取的定性比较的独立方法。在一般情况下，对观察到的社区的影响，发现所有测试参数，虽然珠击和引物的选择有最大的影响。珠击强度的影响与细胞壁强度相关，如通过来自革兰氏阳性细菌的DNA的大幅增加（高达400%）所看到的。然而，同一门内较低的系统发育水平存在显着差异，这表明还有其他因素在起作用。发现基于计算机分析的最佳引物组低估了许多重要的细菌群。对于活性污泥中的16 S rRNA基因分析，我们建议使用FastDNA SPIN试剂盒，其使用4倍于正常珠击和V1-3引物。
DNA extraction and primer choice have a large effect on the observed community structure in all microbial amplicon sequencing analyses. Although the biases are well known, no comprehensive analysis has been conducted in activated sludge communities. In this study we systematically explored the impact of a number of parameters on the observed microbial community: bead beating intensity, primer choice, extracellular DNA removal, and various PCR settings. In total, 176 samples were subjected to 16S rRNA amplicon sequencing, and selected samples were investigated through metagenomics and metatranscriptomics. Quantitative fluorescence in situ hybridization was used as a DNA extraction-independent method for qualitative comparison. In general, an effect on the observed community was found on all parameters tested, although bead beating and primer choice had the largest effect. The effect of bead beating intensity correlated with cell-wall strength as seen by a large increase in DNA from Gram-positive bacteria (up to 400%). However, significant differences were present at lower phylogenetic levels within the same phylum, suggesting that additional factors are at play. The best primer set based on in silico analysis was found to underestimate a number of important bacterial groups. For 16S rRNA gene analysis in activated sludge we recommend using the FastDNA SPIN Kit for Soil with four times the normal bead beating and V1-3 primers.