Influence of DNA Extraction Method, 16S rRNA Targeted Hypervariable Regions, and Sample Origin on Microbial Diversity Detected by 454 Pyrosequencing in Marine Chemosynthetic Ecosystems
Influence of DNA Extraction Method, 16S rRNA Targeted Hypervariable Regions, and Sample Origin on Microbial Diversity Detected by 454 Pyrosequencing in Marine Chemosynthetic Ecosystems
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DOI：
10.1128/aem.00592-14
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发表时间：
2014-08-01
影响因子：
4.4
通讯作者：
Cambon-Bonavita, Marie-Anne
中科院分区：
文献类型：
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作者：
Cruaud, Perrine;Vigneron, Adrien;Cambon-Bonavita, Marie-Anne
下一代测序（NGS）为提高我们对环境微生物多样性的认识开辟了令人兴奋的可能性，允许快速和经济有效地鉴定培养和未培养的微生物。然而，文库制备、测序和结果分析可能提供所研究的群落组成的不准确表示。因此，所有这些步骤都需要认真考虑。在这里，我们评估了DNA提取方法的影响，有针对性的16 S rRNA高变区，和样品来源的454焦磷酸测序在海洋冷泉和热液喷口沉积物中检测到的各种微生物。为了以足够的分类精度分配读段，我们建立了一个数据库，其中包含来自深海海洋沉积物的细菌和细菌的约2,500个序列，根据该领域的参考出版物进行了关联。由于统计和多样性分析以及操作分类单位（OTU）网络的推断，我们表明：（i）虽然DNA提取方法似乎不会影响某些样品的结果，但它们可能会导致其他样品的显着变化;（ii）扩增和测序引物的选择也会显着影响样品中检测到的微生物群落。因此，对于一些微生物谱系，如古细菌ANME-1、ANME-2c和MBG-D和δ变形菌亚群，获得了非常不同比例的焦磷酸测序读数。这项工作清楚地表明，从测序为基础的分析，如焦磷酸测序的结果，应非常仔细地解释。因此，NGS与互补方法的组合，如荧光原位杂交（FISH）/催化报告沉积（CARD）-FISH或定量PCR（Q-PCR），将是理想的，以获得更全面的环境微生物群落的图片。
Next-generation sequencing (NGS) opens up exciting possibilities for improving our knowledge of environmental microbial diversity, allowing rapid and cost-effective identification of both cultivated and uncultivated microorganisms. However, library preparation, sequencing, and analysis of the results can provide inaccurate representations of the studied community compositions. Therefore, all these steps need to be taken into account carefully. Here we evaluated the effects of DNA extraction methods, targeted 16S rRNA hypervariable regions, and sample origins on the diverse microbes detected by 454 pyrosequencing in marine cold seep and hydrothermal vent sediments. To assign the reads with enough taxonomic precision, we built a database with about 2,500 sequences from Archaea and Bacteria from deep-sea marine sediments, affiliated according to reference publications in the field. Thanks to statistical and diversity analyses as well as inference of operational taxonomic unit (OTU) networks, we show that (i) while DNA extraction methods do not seem to affect the results for some samples, they can lead to dramatic changes for others; and (ii) the choice of amplification and sequencing primers also considerably affects the microbial community detected in the samples. Thereby, very different proportions of pyrosequencing reads were obtained for some microbial lineages, such as the archaeal ANME-1, ANME-2c, and MBG-D and deltaproteobacterial subgroups. This work clearly indicates that the results from sequencing-based analyses, such as pyrosequencing, should be interpreted very carefully. Therefore, the combination of NGS with complementary approaches, such as fluorescence in situ hybridization (FISH)/catalyzed reporter deposition (CARD)-FISH or quantitative PCR (Q-PCR), would be desirable to gain a more comprehensive picture of environmental microbial communities.