High-throughput sequencing of 16S rRNA gene amplicons: effects of extraction procedure, primer length and annealing temperature.
High-throughput sequencing of 16S rRNA gene amplicons: effects of extraction procedure, primer length and annealing temperature.
复制标题
DOI：
10.1371/journal.pone.0038094
复制
发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Pallen MJ
中科院分区：
文献类型：
--
作者：
Sergeant MJ;Constantinidou C;Cogan T;Penn CW;Pallen MJ
分析16S-rDNA序列以评估样本的细菌群落组成是一种广泛使用的技术，随着高通量测序的出现而增加。虽然人们已经致力于确定16S基因信息量最大的区域和处理数据的最佳信息学程序，但对PCR步骤，特别是退火温度和引物长度的关注很少。为解决这一问题，从鸡盲肠内容物DNA中扩增出16S-rDNA，采用不同的退火温度、不同的引物和不同的DNA提取程序。对扩增产物进行焦解测序，以确定从鸡盲肠样本中捕获最大细菌多样性的最佳方案。即使在非常低的温度下，对群落结构的影响也很小，尽管一些OTU如双歧杆菌的丰度增加了。使用较短的引物没有揭示任何新的OTUS，但确实改变了所获得的社区概况。通过打珠对样品进行机械破碎对获得的结果有显著影响，反复冻结和解冻也是如此。总而言之，现有的引物和标准的退火温度与较低的退火温度和较短的引物获得了同样多的多样性。
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.
登录
查看更多内容
影响因子：
5.6
作者：
Lutful Kabir SM
通讯作者：
Lutful Kabir SM
影响因子：
4.4
作者：
Scupham, A. J.;Jones, J. A.;Weber, T. E.
通讯作者：
Weber, T. E.
影响因子：
14.9
作者：
Cole JR;Wang Q;Cardenas E;Fish J;Chai B;Farris RJ;Kulam-Syed-Mohideen AS;McGarrell DM;Marsh T;Garrity GM;Tiedje JM
通讯作者：
Tiedje JM
影响因子：
14.9
作者：
Claesson MJ;Wang Q;O'Sullivan O;Greene-Diniz R;Cole JR;Ross RP;O'Toole PW
通讯作者：
O'Toole PW
影响因子：
4.4
作者：
Bru, D.;Martin-Laurent, F.;Philippot, L.
通讯作者：
Philippot, L.