Hanning Irene B, Ricke Steven C
Department of Food Science and Technology, University of Tennessee, Knoxville, TN, USA.
Methods Mol Biol. 2011;733:159-70. doi: 10.1007/978-1-61779-089-8_11.
Next-generation sequencing (NGS) is a powerful tool that can be utilized to profile and compare microbial populations. By amplifying a target gene present in all bacteria and subsequently sequencing amplicons, the bacteria genera present in the populations can be identified and compared. In some scenarios, little to no difference may exist among microbial populations being compared in which case a prescreening method would be practical to determine which microbial populations would be suitable for further analysis by NGS. Denaturing density-gradient electrophoresis (DGGE) is relatively cheaper than NGS and the data comparing microbial populations are ready to be viewed immediately after electrophoresis. DGGE follows essentially the same initial methodology as NGS by targeting and amplifying the 16S rRNA gene. However, as opposed to sequencing amplicons, DGGE amplicons are analyzed by electrophoresis. By prescreening microbial populations with DGGE, more efficient use of NGS methods can be accomplished. In this chapter, we outline the protocol for DGGE targeting the same gene (16S rRNA) that would be targeted for NGS to compare and determine differences in microbial populations from a wide range of ecosystems.
下一代测序（NGS）是一种强大的工具，可用于分析和比较微生物群落。通过扩增所有细菌中都存在的靶基因，随后对扩增子进行测序，可以识别和比较群落中存在的细菌属。在某些情况下，所比较的微生物群落之间可能几乎没有差异，在这种情况下，预筛选方法将有助于确定哪些微生物群落适合通过NGS进行进一步分析。变性梯度凝胶电泳（DGGE）比NGS相对便宜，并且比较微生物群落的数据在电泳后即可立即查看。DGGE通过靶向和扩增16S rRNA基因，基本上遵循与NGS相同的初始方法。然而，与对扩增子进行测序不同，DGGE扩增子是通过电泳进行分析的。通过用DGGE对微生物群落进行预筛选，可以更有效地使用NGS方法。在本章中，我们概述了针对与NGS相同基因（16S rRNA）的DGGE方案，以比较和确定来自广泛生态系统的微生物群落的差异。
