Optimizing the analysis of human intestinal microbiota with phylogenetic microarray.
Optimizing the analysis of human intestinal microbiota with phylogenetic microarray.
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DOI：
10.1111/j.1574-6941.2010.01009.x
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发表时间：
2011-02
影响因子：
4.2
通讯作者：
Paliy O
中科院分区：
文献类型：
--
作者：
Rigsbee L;Agans R;Foy BD;Paliy O
系统发育微阵列为复杂微生物群落的高通量分析提供了一种有吸引力的策略。在这项工作中，我们提出了几种方法来优化肠道微生物群分析与最近开发的微生物群阵列。首先，我们确定了16S rdna特异性PCR扩增如何影响细菌检测和测量丰度值的一致性。细菌检测随着PCR扩增周期的增加而提高，但25个周期足以达到最大可能的检测。还观察到pcr引起的丰度测量值偏差。我们还开发了两种数学算法，旨在解释不同物种之间16S rRNA片段的交叉杂交预测，并根据每个物种基因组的16S rRNA基因拷贝数调整测量的杂交信号。16S rRNA基因拷贝调整表明，在一些基于16S rRNA的研究中，梭状芽孢杆菌类成员的存在可能被高估了。最后，我们证明了用系统发育微阵列检测总群落RNA可以提供个体群落成员相对代谢活性的估计。从同一样品中分离的基因组DNA和总RNA的互补分析提供了评估同一微生物群落中种群结构和活动的机会。
Phylogenetic microarrays present an attractive strategy to high-throughput interrogation of complex microbial communities. In this work we present several approaches to optimize the analysis of intestinal microbiota with the recently developed Microbiota Array. First, we determined how 16S rDNA-specific PCR amplification influenced bacterial detection and the consistency of measured abundance values. Bacterial detection improved with an increase in the number of PCR amplification cycles, but 25 cycles were sufficient to achieve the maximum possible detection. A PCR-caused deviation in the measured abundance values was also observed. We also developed two mathematical algorithms aimed to account for a predicted cross-hybridization of 16S rDNA fragments among different species, and to adjust the measured hybridization signal based on the number of 16S rRNA gene copies per species genome. The 16S rRNA gene copy adjustment indicated that the presence of members of class Clostridia might be over-estimated in some 16S rDNA-based studies. Finally, we show that the examination of total community RNA with phylogenetic microarray can provide estimates of the relative metabolic activity of individual community members. Complementary profiling of genomic DNA and total RNA isolated from the same sample presents an opportunity to assess population structure and activity in the same microbial community.
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DOI：
10.1073/pnas.0608255104
发表时间：
2007-01-02
影响因子：
11.1
作者：
Brodie, Eoin L.;DeSantis, Todd Z.;Andersen, Gary L.
通讯作者：
Andersen, Gary L.
影响因子：
56.9
作者：
Gill, Steven R.;Pop, Mihai;Nelson, Karen E.
通讯作者：
Nelson, Karen E.
影响因子：
4.4
作者：
Acinas, SG;Sarma-Rupavtarm, R;Polz, MF
通讯作者：
Polz, MF
影响因子：
5.1
作者：
Scanlan, Pauline D.;Shanahan, Fergus;Marchesi, Julian R.
通讯作者：
Marchesi, Julian R.
影响因子：
3.2
作者：
Gyaneshwar, P;Paliy, O;Kustu, S
通讯作者：
Kustu, S