Analysis, optimization and verification of Illumina-generated 16S rRNA gene amplicon surveys.
Analysis, optimization and verification of Illumina-generated 16S rRNA gene amplicon surveys.
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DOI：
10.1371/journal.pone.0094249
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Graf J
中科院分区：
文献类型：
--
作者：
Nelson MC;Morrison HG;Benjamino J;Grim SL;Graf J
利用低成本、高通量的测序仪器，通过16S rRNA基因测序来探索微生物群落。基于illumina的16S rRNA基因测序由于其更低的成本、更高的准确性和更高的通量，最近比454焦磷酸测序更受欢迎。尽管最近的报道表明Illumina和454焦磷酸测序提供了相似的β多样性测量，但仍有待证明，通过简单地改变引物的测序适配器，现有的454焦磷酸测序工作流程可以直接从454转移到Illumina MiSeq测序。在这项研究中，我们修改了454个焦磷酸测序引物，靶向16S rRNA基因的V4-V5高变区，使其与Illumina测序仪兼容。对奶牛、人、水蛭、小鼠、污水、白蚁和模拟群落的微生物群落进行了V4- v5区和V4区454和MiSeq测序。我们的分析表明，与从头聚类相比，单独基于参考的OTU聚类引入了偏差，导致某些样本中无法观察到某些分类群。在此基础上，我们设计并推荐了一个分析管道，包括读取合并、污染物过滤和基于参考的聚类，然后是从头开始的OTU聚类，从而产生与从头开始的OTU聚类分析一致的多样性度量。发现Illumina测序的低水平数据集污染可能影响需要高灵敏度方法的分析。虽然迁移到基于illumina的测序平台有望对微生物多样性的广度和功能提供更深入的见解，但我们的研究结果表明，必须注意确保测序和处理工件不会掩盖真正的微生物多样性。
The exploration of microbial communities by sequencing 16S rRNA genes has expanded with low-cost, high-throughput sequencing instruments. Illumina-based 16S rRNA gene sequencing has recently gained popularity over 454 pyrosequencing due to its lower costs, higher accuracy and greater throughput. Although recent reports suggest that Illumina and 454 pyrosequencing provide similar beta diversity measures, it remains to be demonstrated that pre-existing 454 pyrosequencing workflows can transfer directly from 454 to Illumina MiSeq sequencing by simply changing the sequencing adapters of the primers. In this study, we modified 454 pyrosequencing primers targeting the V4-V5 hyper-variable regions of the 16S rRNA gene to be compatible with Illumina sequencers. Microbial communities from cows, humans, leeches, mice, sewage, and termites and a mock community were analyzed by 454 and MiSeq sequencing of the V4-V5 region and MiSeq sequencing of the V4 region. Our analysis revealed that reference-based OTU clustering alone introduced biases compared to de novo clustering, preventing certain taxa from being observed in some samples. Based on this we devised and recommend an analysis pipeline that includes read merging, contaminant filtering, and reference-based clustering followed by de novo OTU clustering, which produces diversity measures consistent with de novo OTU clustering analysis. Low levels of dataset contamination with Illumina sequencing were discovered that could affect analyses that require highly sensitive approaches. While moving to Illumina-based sequencing platforms promises to provide deeper insights into the breadth and function of microbial diversity, our results show that care must be taken to ensure that sequencing and processing artifacts do not obscure true microbial diversity.
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影响因子：
12.3
作者：
Huse SM;Huber JA;Morrison HG;Sogin ML;Welch DM
通讯作者：
Welch DM
DOI：
10.1038/ismej.2012.8
发表时间：
2012-08
期刊：
The ISME journal
影响因子：
--
作者：
通讯作者：
--
影响因子：
11.4
作者：
Nelson, Michael C.;Morrison, Mark;Yu, Zhongtang
通讯作者：
Yu, Zhongtang
影响因子：
46.9
作者：
Loman, Nicholas J.;Misra, Raju V.;Pallen, Mark J.
通讯作者：
Pallen, Mark J.
影响因子：
4.4
作者：
Bartram, Andrea K.;Lynch, Michael D. J.;Neufeld, Josh D.
通讯作者：
Neufeld, Josh D.