Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons
Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons
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DOI：
10.1101/gr.112730.110
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发表时间：
2011-03-01
期刊：
影响因子：
7
通讯作者：
Birren, Bruce W.
中科院分区：
文献类型：
--
作者：
Haas, Brian J.;Gevers, Dirk;Birren, Bruce W.
环境样本中的细菌多样性通常通过PCR扩增的16 S rRNA基因（16 S）序列来评估。然而，感知的多样性可能受到样品制备、引物选择和嵌合16S扩增产物形成的影响。嵌合体是多个亲本序列之间的杂交产物，可以被错误地解释为新的生物体，从而扩大了明显的多样性。我们开发了一种新的嵌合体检测工具，称为嵌合体杀手（CS）。CS检测嵌合体的灵敏度比以前的方法更高，在短序列上表现良好，例如由454生命科学（罗氏）基因组测序仪产生的序列，并且可以扩展到大型数据集。通过基准CS性能对来自已知生物体和模拟嵌合体集的受控DNA混合物的序列，我们提供了影响嵌合体形成的因素，如序列丰度，16S基因之间的相似性程度和PCR条件的见解。嵌合体被发现可重复形成独立的扩增，并有助于错误的看法，样品的多样性和错误的识别新的类群，与丰富的物种表现出嵌合率超过70%。我们的模拟社区的鸟枪宏基因组序列似乎没有16S嵌合体，支持鸟枪宏基因组学在验证靶向序列调查中发现的新生物体中的作用。
Bacterial diversity among environmental samples is commonly assessed with PCR-amplified 16S rRNA gene (16S) sequences. Perceived diversity, however, can be influenced by sample preparation, primer selection, and formation of chimeric 16S amplification products. Chimeras are hybrid products between multiple parent sequences that can be falsely interpreted as novel organisms, thus inflating apparent diversity. We developed a new chimera detection tool called Chimera Slayer (CS). CS detects chimeras with greater sensitivity than previous methods, performs well on short sequences such as those produced by the 454 Life Sciences (Roche) Genome Sequencer, and can scale to large data sets. By benchmarking CS performance against sequences derived from a controlled DNA mixture of known organisms and a simulated chimera set, we provide insights into the factors that affect chimera formation such as sequence abundance, the extent of similarity between 16S genes, and PCR conditions. Chimeras were found to reproducibly form among independent amplifications and contributed to false perceptions of sample diversity and the false identification of novel taxa, with less-abundant species exhibiting chimera rates exceeding 70%. Shotgun metagenomic sequences of our mock community appear to be devoid of 16S chimeras, supporting a role for shotgun metagenomics in validating novel organisms discovered in targeted sequence surveys.