Department of Environmental Science and Policy, George Mason University, Manassas, Virginia, USA.
Biotechniques. 2012 Dec;53(6):381-3. doi: 10.2144/000113967.
Multi-tag pyrosequencing has become a key method in the analysis of microbial community composition. However, it is well known that kinetic bias during the initial PCR amplification of such microbial communities can dramatically distort amplicon abundance prior to downstream emulsion PCR and pyrosequencing. Here we present a simple protocol combining length-heterogeneity PCR fingerprinting with pyrosequencing to ensure the linearity of microbial community amplification. The method employs a fluorescently labeled reverse primer along with multi-tagged forward primers to initially amplify the microbial community. The resulting labeled amplicons are then fingerprinted, purified, and quantitated prior to emulsion PCR and pyrosequencing. Our data demonstrates: (i) use of this protocol results in a distribution of sequences showing linear amplification following emulsion PCR when compared with the initial length-heterogeneity PCR fingerprints, and (ii) that the added tags and labels do not have a negative effect on overall microbial community profiles.
多重标签焦磷酸测序已成为分析微生物群落组成的关键方法。然而，众所周知，在对这种微生物群落进行初始 PCR 扩增时，动力学偏差会在下游乳液 PCR 和焦磷酸测序之前极大地扭曲扩增子丰度。在这里，我们提出了一种简单的方案，将长度异质性 PCR 指纹图谱与焦磷酸测序相结合，以确保微生物群落扩增的线性。该方法使用荧光标记的反向引物和多标签正向引物来初始扩增微生物群落。然后对标记的扩增子进行指纹图谱分析、纯化和定量，然后进行乳液 PCR 和焦磷酸测序。我们的数据表明：（i）与初始长度异质性 PCR 指纹图谱相比，使用该方案进行乳液 PCR 后，得到的序列分布显示出线性扩增；（ii）添加的标签和标记对整个微生物群落图谱没有负面影响。
