Centre for Systems Biology, School of Biosciences, University of Birmingham, Birmingham, United Kingdom.
PLoS One. 2012;7(5):e38094. doi: 10.1371/journal.pone.0038094. Epub 2012 May 29.
The analysis of 16S-rDNA sequences to assess the bacterial community composition of a sample is a widely used technique that has increased with the advent of high throughput sequencing. Although considerable effort has been devoted to identifying the most informative region of the 16S gene and the optimal informatics procedures to process the data, little attention has been paid to the PCR step, in particular annealing temperature and primer length. To address this, amplicons derived from 16S-rDNA were generated from chicken caecal content DNA using different annealing temperatures, primers and different DNA extraction procedures. The amplicons were pyrosequenced to determine the optimal protocols for capture of maximum bacterial diversity from a chicken caecal sample. Even at very low annealing temperatures there was little effect on the community structure, although the abundance of some OTUs such as Bifidobacterium increased. Using shorter primers did not reveal any novel OTUs but did change the community profile obtained. Mechanical disruption of the sample by bead beating had a significant effect on the results obtained, as did repeated freezing and thawing. In conclusion, existing primers and standard annealing temperatures captured as much diversity as lower annealing temperatures and shorter primers.
采用 16S-rDNA 序列分析来评估样本中的细菌群落组成是一种广泛应用的技术，随着高通量测序的出现，该技术得到了进一步发展。尽管人们已经投入了相当大的精力来确定 16S 基因中最具信息量的区域和处理数据的最佳信息学程序，但对于 PCR 步骤，特别是退火温度和引物长度，关注较少。为了解决这个问题，我们使用不同的退火温度、引物和不同的 DNA 提取程序，从鸡盲肠内容物 DNA 中扩增 16S-rDNA 片段。对扩增子进行焦磷酸测序，以确定从鸡盲肠样本中捕获最大细菌多样性的最佳方案。即使在非常低的退火温度下，对群落结构的影响也很小，尽管某些 OTUs（如双歧杆菌）的丰度增加了。使用较短的引物并没有发现任何新的 OTUs，但确实改变了获得的群落图谱。通过珠磨法机械破坏样品对结果有显著影响，反复冻融也有显著影响。总之，现有的引物和标准退火温度与较低的退火温度和较短的引物一样，能够捕获到更多的多样性。
