PCR-induced sequence artifacts and bias: Insights from comparison of two 16S rRNA clone libraries constructed from the same sample
PCR-induced sequence artifacts and bias: Insights from comparison of two 16S rRNA clone libraries constructed from the same sample
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DOI：
10.1128/aem.71.12.8966-8969.2005
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发表时间：
2005-12-01
影响因子：
4.4
通讯作者：
Polz, MF
中科院分区：
文献类型：
--
作者：
Acinas, SG;Sarma-Rupavtarm, R;Polz, MF
从一个复杂的浮游细菌样品的16S rRNA基因序列多样性的PCR文物的贡献进行了估计。Taq DNA聚合酶错误被认为是占主导地位的序列伪影，但可以通过将序列聚类为99%的序列相似性组来限制。其他人为因素（嵌合体和异源双链分子）显着减少采用修改后的扩增方案。令人惊讶的是，在由扩增不同循环数的PCR产物构建的两个文库中未检测到序列类型的偏斜。建议修改的扩增方案和报告的多样性估计在99%的序列相似性作为一个标准。
The contribution of PCR artifacts to 16S rRNA gene sequence diversity from a complex bacterioplankton sample was estimated. Taq DNA polymerase errors were found to be the dominant sequence artifact but could be constrained by clustering the sequences into 99% sequence similarity groups. Other artifacts (chimeras and heteroduplex molecules) were significantly reduced by employing modified amplification protocols. Surprisingly, no skew in sequence types was detected in the two libraries constructed from PCR products amplified for different numbers of cycles. Recommendations for modification of amplification protocols and for reporting diversity estimates at 99% sequence similarity as a standard are given.