Corcoll Natàlia, Österlund Tobias, Sinclair Lucas, Eiler Alexander, Kristiansson Erik, Backhaus Thomas, Eriksson K Martin
Department of Biological and Environmental Sciences, University of Gothenburg, SE-405 30 Gothenburg, Sweden.
Department of Mathematical Sciences, Chalmers University of Technology, SE-41296 Gothenburg, Sweden.
FEMS Microbiol Lett. 2017 Aug 1;364(14). doi: 10.1093/femsle/fnx139.
High-throughput DNA sequencing technologies are increasingly used for the metagenomic characterisation of microbial biodiversity. However, basic issues, such as the choice of an appropriate DNA extraction method, are still not resolved for non-model microbial communities. This study evaluates four commonly used DNA extraction methods for marine periphyton biofilms in terms of DNA yield, efficiency, purity, integrity and resulting 16S rRNA bacterial diversity. Among the tested methods, the Plant DNAzol® Reagent (PlantDNAzol) and the FastDNA® SPIN Kit for Soil (FastDNA Soil) methods were best suited to extract high quantities of DNA (77-130 μg g wet wt-1). Lower amounts of DNA were obtained (<37 μg g wet wt-1) with the Power Plant® Pro DNA Isolation Kit (PowerPlant) and the Power Biofilm® DNA Isolation Kit (PowerBiofilm) methods, but integrity and purity of the extracted DNA were higher. Results from 16S rRNA amplicon sequencing demonstrate that the choice of a DNA extraction method significantly influences the bacterial community profiles generated. A higher number of bacterial OTUs were detected when DNA was extracted with the PowerBiofilm and the PlantDNAzol methods. Overall, this study demonstrates the potential bias in metagenomic diversity estimates associated with different DNA extraction methods.
高通量DNA测序技术越来越多地用于微生物多样性的宏基因组学表征。然而，对于非模式微生物群落，诸如选择合适的DNA提取方法等基本问题仍未得到解决。本研究从DNA产量、效率、纯度、完整性以及由此产生的16S rRNA细菌多样性方面，评估了四种常用于海洋附生生物膜的DNA提取方法。在所测试的方法中，植物DNAzol试剂（PlantDNAzol）和土壤快速DNA SPIN试剂盒（FastDNA Soil）方法最适合提取大量DNA（77 - 130μg g湿重-1）。使用植物动力Pro DNA分离试剂盒（PowerPlant）和生物膜动力DNA分离试剂盒（PowerBiofilm）方法获得的DNA量较低（<37μg g湿重-1），但提取的DNA完整性和纯度较高。16S rRNA扩增子测序结果表明，DNA提取方法的选择会显著影响所产生的细菌群落概况。当使用PowerBiofilm和PlantDNAzol方法提取DNA时，检测到的细菌OTU数量更多。总体而言，本研究证明了与不同DNA提取方法相关的宏基因组多样性估计中的潜在偏差。
