Illumina-based analysis of microbial community diversity
Illumina-based analysis of microbial community diversity
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DOI：
10.1038/ismej.2011.74
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发表时间：
2012-01-01
期刊：
影响因子：
11
通讯作者：
Ochman, Howard
中科院分区：
文献类型：
--
作者：
Degnan, Patrick H.;Ochman, Howard
微生物通常存在于不同复杂性和多样性的环境中。尽管基于培养的技术无法准确捕获微生物群落的真正多样性，但这些缺陷已经通过应用针对普遍保守的16S核糖体RNA基因的分子方法来克服。最近应用454焦磷酸测序同时测序数千个16S rDNA序列（pyrotags）已经彻底改变了复杂微生物群落的表征。迄今为止，基于454 pyrotag的研究主导了该领域，但已经开发出以更低成本产生更多序列读取的测序平台。在这里，我们使用Illumina测序平台设计了一种16S扩增子分析（iTags）策略，并评估了其通用性、实用性和潜在的复杂性。我们制作并测序了从含量不同的样品中扩增的高可变16S rDNA片段的配对端文库，从单个细菌到高度复杂的群落。我们采用了一种方法，允许我们评估几个潜在的错误来源，包括测序伪影、扩增偏差、不对应的对端读取和分类分类错误。通过考虑每个错误来源，我们描述了从数百万个测序读数中获得生物学相关和可靠结论的方法，这些测序读数可以很容易地通过该技术生成。ISME Journal (2012) 6, 183-194；doi: 10.1038 / ismej.2011.74;2011年6月16日在线发布
Microbes commonly exist in milieus of varying complexity and diversity. Although cultivation-based techniques have been unable to accurately capture the true diversity within microbial communities, these deficiencies have been overcome by applying molecular approaches that target the universally conserved 16S ribosomal RNA gene. The recent application of 454 pyrosequencing to simultaneously sequence thousands of 16S rDNA sequences (pyrotags) has revolutionized the characterization of complex microbial communities. To date, studies based on 454 pyrotags have dominated the field, but sequencing platforms that generate many more sequence reads at much lower costs have been developed. Here, we use the Illumina sequencing platform to design a strategy for 16S amplicon analysis (iTags), and assess its generality, practicality and potential complications. We fabricated and sequenced paired-end libraries of amplified hyper-variable 16S rDNA fragments from sets of samples that varied in their contents, ranging from a single bacterium to highly complex communities. We adopted an approach that allowed us to evaluate several potential sources of errors, including sequencing artifacts, amplification biases, non-corresponding paired-end reads and mistakes in taxonomic classification. By considering each source of error, we delineate ways to make biologically relevant and robust conclusions from the millions of sequencing reads that can be readily generated by this technology. The ISME Journal (2012) 6, 183-194; doi:10.1038/ismej.2011.74; published online 16 June 2011