Janabi Ali H D, Kerkhof Lee J, McGuinness Lora R, Biddle Amy S, McKeever Kenneth H
Microbial Biology Graduate Program, Rutgers-The State University of New Jersey, New Brunswick, NJ 08901, USA.
Department of Marine and Coastal Sciences, Rutgers-The State University of New Jersey, New Brunswick, NJ 08901, USA.
J Microbiol Methods. 2016 Oct;129:14-19. doi: 10.1016/j.mimet.2016.07.019. Epub 2016 Jul 25.
There are many choices for methods of extracting bacterial DNA for Next Generation Sequencing (NGS) from fecal samples. Here, we compare our modifications of a phenol/chloroform extraction method plus an inhibitor removal solution (C3) (ph/Chl+C3) to the PowerFecal® DNA Isolation Kit (MoBio-K). DNA quality and quantity coupled to NGS results were used to assess differences in relative abundance, Shannon diversity index, unique species, and principle coordinate analysis (PCoA) between biological replicates. Six replicate samples, taken from a single ball of horse feces manually collected from the rectum, were subjected to each extraction method. The Ph/Chl+C3 method produced 100× higher DNA yields with less shearing than the MoBio-K method. To assess the methods, the two method samples were sent for sequencing of the bacterial V3-V4 region of 16S rRNA gene using the Illumina MiSeq platform. The relative abundance of Bacteroidetes was greater and there were more unique species assigned to this group in MoBio-K than in Ph/Chl+C3 (P<0.05). In contrast, Firmicutes had greater relative abundance and more unique species in Ph/Chl+C3 extracts than in MoBio-K (P<0.05). The other major bacterial phyla were equally abundant in samples using both extraction methods. Alpha diversity and Shannon Weaver indices showed greater evenness of bacterial distribution in Ph/Chl+C3 compared with MoBio-K (P<0.05), but there was no difference in the OTU richness. Principle coordinate analysis (PCoA) indicated a distinct separation between the two methods (P<0.05) and tighter clustering (less variability) in Ph/Chl+C3 than in MoBio-K. These results suggest that the Ph/Chl+C3 may be preferred for research to identify specific Firmicutes taxa such as Clostridium, and Bacillus. However; MoBio-K may be a better choice for projects focusing on Bacteroidetes abundance. The Ph/Chl+C3 method required less time, but has some safety concerns associated with exposure and disposal of phenol and chloroform. While the MoBio-K may be better choice for researchers with less access to safety equipment like a fume hood.
从粪便样本中提取用于下一代测序（NGS）的细菌DNA的方法有很多种。在此，我们将改良的酚/氯仿提取法加抑制剂去除溶液（C3）（ph/Chl+C3）与PowerFecal® DNA分离试剂盒（MoBio-K）进行比较。结合NGS结果的DNA质量和数量用于评估生物重复样本之间在相对丰度、香农多样性指数、独特物种以及主坐标分析（PCoA）方面的差异。从直肠手动采集的一个马粪球中取出六个重复样本，对每个样本进行每种提取方法的处理。与MoBio-K方法相比，Ph/Chl+C3方法产生的DNA产量高100倍，且剪切较少。为了评估这些方法，将两种方法处理的样本送去使用Illumina MiSeq平台对16S rRNA基因的细菌V3-V4区域进行测序。在MoBio-K方法中，拟杆菌门的相对丰度更高，且该组中分配到的独特物种比Ph/Chl+C3方法更多（P<0.05）。相反，在Ph/Chl+C3提取物中，厚壁菌门的相对丰度更高，且独特物种比MoBio-K方法更多（P<0.05）。使用两种提取方法的样本中，其他主要细菌门的丰度相同。α多样性和香农-韦弗指数表明，与MoBio-K相比，Ph/Chl+C3中细菌分布的均匀度更高（P<0.05），但OTU丰富度没有差异。主坐标分析（PCoA）表明两种方法之间存在明显分离（P<0.05），且Ph/Chl+C3中的聚类比MoBio-K更紧密（变异性更小）。这些结果表明，对于识别特定厚壁菌门分类群（如梭菌属和芽孢杆菌属）的研究，Ph/Chl+C3可能更受青睐。然而，对于关注拟杆菌门丰度的项目，MoBio-K可能是更好的选择。Ph/Chl+C3方法所需时间更少，但存在与酚和氯仿的接触及处置相关的一些安全问题。而对于较难获得通风橱等安全设备的研究人员来说，MoBio-K可能是更好的选择。
