Mathematical modeling of 16S ribosomal DNA amplification reveals optimal conditions for the interrogation of complex microbial communities with phylogenetic microarrays
Mathematical modeling of 16S ribosomal DNA amplification reveals optimal conditions for the interrogation of complex microbial communities with phylogenetic microarrays
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DOI：
10.1093/bioinformatics/btr326
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发表时间：
2011-08-01
期刊：
影响因子：
5.8
通讯作者：
Foy, Brent D.
中科院分区：
文献类型：
--
作者：
Paliy, Oleg;Foy, Brent D.
动机：目前对复杂微生物群落的许多研究依赖于群落基因组DNA的分离、16S核糖体RNA基因(RDNA)的扩增以及随后通过高通量测序、系统发育微阵列或定量PCR询问扩增的16S rDNA池来检查群落结构。结果：本文描述了一个数学模型的建立，旨在模拟16S核糖体DNA样本的多模板扩增，以及随后通过系统发育微阵列对这些扩增的16S rDNA物种的检测。利用微生物区系阵列在肠道微生物群落分析中获得的实验结果估计的参数，我们表明物种检测和物种丰度估计的准确性在很大程度上取决于用于扩增16S rDNA的PCR循环的数量。这两个参数最初都随着每个额外的PCR循环而改善，并在扩增15到20个循环之间达到最佳。然而，使用20多个循环的PCR扩增和/或超过50 ng的起始基因组DNA模板，既不利于检测到的群落成员的比例，也不利于丰度估计的准确性。总体而言，模型模拟的结果与可用的实验数据很好地匹配。我们的模拟还表明，物种检测和丰度测量的准确性与较高的样本范围的聚合酶链式反应扩增率、较低的模板对模板聚合酶链式反应偏倚和询问群落中较少的物种数量呈正相关。开发的模型可以很容易地修改以模拟其他多模板DNA混合物以及其他微阵列设计和PCR扩增方案。
Motivation: Many current studies of complex microbial communities rely on the isolation of community genomic DNA, amplification of 16S ribosomal RNA genes (rDNA) and subsequent examination of community structure through interrogation of the amplified 16S rDNA pool by high-throughput sequencing, phylogenetic microarrays or quantitative PCR.Results: Here we describe the development of a mathematical model aimed to simulate multitemplate amplification of 16S ribosomal DNA sample and subsequent detection of these amplified 16S rDNA species by phylogenetic microarray. Using parameters estimated from the experimental results obtained in the analysis of intestinal microbial communities with Microbiota Array, we show that both species detection and the accuracy of species abundance estimates depended heavily on the number of PCR cycles used to amplify 16S rDNA. Both parameters initially improved with each additional PCR cycle and reached optimum between 15 and 20 cycles of amplification. The use of more than 20 cycles of PCR amplification and/or more than 50 ng of starting genomic DNA template was, however, detrimental to both the fraction of detected community members and the accuracy of abundance estimates. Overall, the outcomes of the model simulations matched well available experimental data. Our simulations also showed that species detection and the accuracy of abundance measurements correlated positively with the higher sample-wide PCR amplification rate, lower template-to-template PCR bias and lower number of species in the interrogated community. The developed model can be easily modified to simulate other multitemplate DNA mixtures as well as other microarray designs and PCR amplification protocols.