Effects of polymerase, template dilution and cycle number on PCR based 16 S rRNA diversity analysis using the deep sequencing method.
Effects of polymerase, template dilution and cycle number on PCR based 16 S rRNA diversity analysis using the deep sequencing method.
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DOI：
10.1186/1471-2180-10-255
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发表时间：
2010-10-12
期刊：
影响因子：
4.2
通讯作者：
Zhou HW
中科院分区：
文献类型：
--
作者：
Wu JY;Jiang XT;Jiang YX;Lu SY;Zou F;Zhou HW
已发现引物和扩增子长度影响通过焦磷酸测序进行的基于PCR的微生物多样性估计，而其他PCR条件尚未使用任何深度测序方法解决。本研究使用Solexa平台确定了聚合酶、模板稀释度和PCR循环次数的影响。具有较高保真度的PfuUltra II Fusion HS DNA聚合酶（Stratagene）显示出较低量的PCR伪影，并确定了比Ex Taq（Takara）更低的分类群丰富度。更重要的是，这两种聚合酶在扩增一些非常丰富的序列时表现出不同的效率，并决定了显着不同的群落结构。正如预期的那样，DNA模板的稀释导致分类群丰富度的估计降低，特别是在200倍稀释水平下，但所有稀释水平的群落结构相似。与25个循环组相比，30个循环组增加了PCR伪影，但测定的类群丰富度低于25个循环组。PCR循环次数对微生物群落结构没有显著影响。这些结果突出了PCR条件，特别是聚合酶，对下一代测序方法进行微生物多样性分析具有显著影响。
The primer and amplicon length have been found to affect PCR based estimates of microbial diversity by pyrosequencing, while other PCR conditions have not been addressed using any deep sequencing method. The present study determined the effects of polymerase, template dilution and PCR cycle number using the Solexa platform. The PfuUltra II Fusion HS DNA Polymerase (Stratagene) with higher fidelity showed lower amount of PCR artifacts and determined lower taxa richness than the Ex Taq (Takara). More importantly, the two polymerases showed different efficiencies for amplifying some of very abundant sequences, and determined significantly different community structures. As expected, the dilution of the DNA template resulted in a reduced estimation of taxa richness, particularly at the 200 fold dilution level, but the community structures were similar for all dilution levels. The 30 cycle group increased the PCR artifacts while comparing to the 25 cycle group, but the determined taxa richness was lower than that of the 25 cycle group. The PCR cycle number did not changed the microbial community structure significantly. These results highlight the PCR conditions, particularly the polymerase, have significant effect on the analysis of microbial diversity with next generation sequencing methods.
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