Development of a genus-specific next generation sequencing approach for sensitive and quantitative determination of the Legionella microbiome in freshwater systems.
Development of a genus-specific next generation sequencing approach for sensitive and quantitative determination of the Legionella microbiome in freshwater systems.
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DOI：
10.1186/s12866-017-0987-5
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发表时间：
2017-03-31
期刊：
影响因子：
4.2
通讯作者：
Höfle MG
中科院分区：
文献类型：
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作者：
Pereira RP;Peplies J;Brettar I;Höfle MG
下一代测序（NGS）通过在基于PCR的方法中使用细菌通用引物靶向16 S rRNA基因，彻底改变了天然和人造微生物群落的分析。在我们的研究中，我们将引物特异性缩小到单一的单系属，因为对于微生物学中的许多问题，只有整个微生物组的特定部分是感兴趣的。我们选择了军团菌属，包括20多个致病物种，因为它与水基呼吸道感染高度相关。通过使用Illumina MiSeq技术对军团菌属特异性的16 S rRNA基因扩增子进行测序，设计了一种新的基于NGS的方法。这种方法进行了验证，并适用于一组有代表性的淡水样品。我们的研究结果显示，生成的文库呈现出较低的每碱基平均原始错误率（<0.5%）;并证实了高保真酶（如KAPA HiFi）的使用，以提高序列准确性和质量。该方法还显示出高的原位特异性（>95%）和非常好的可重复性。仅在存在γ细菌分支SAR 86的样品中观察到超过1%的非军团菌序列。下一代测序读段计数没有显示出相当大的扩增/测序偏倚，并且显示出对L.嗜肺沿着稀释范围使用加标的经认证的基因组标准品测定。基因组标准品和由六种不同军团菌组成的模拟群落表明，所开发的NGS方法在单个物种水平上是定量和特异性的，包括L。嗜肺菌与通用NGS方法相比，我们的属特异性方法的灵敏度至少高一个数量级。通过实时PCR定量的比较显示与NGS数据一致。总体而言，我们的NGS方法可以确定军团菌属的数量丰度，即。e.完整的军团菌微生物组，而不需要物种特异性引物。开发的NGS方法提供了一种新的分子监测工具，如果使用加标的基因组标准品来校准该方法，则可以在定性和定量方面监测所有军团菌属。总的来说，属特异性NGS方法以定量、特异和敏感的方式为大规模并行诊断开辟了一条新途径。本文的在线版本（doi：10.1186/s12866-017-0987-5）包含补充材料，可供授权用户使用。
Next Generation Sequencing (NGS) has revolutionized the analysis of natural and man-made microbial communities by using universal primers for bacteria in a PCR based approach targeting the 16S rRNA gene. In our study we narrowed primer specificity to a single, monophyletic genus because for many questions in microbiology only a specific part of the whole microbiome is of interest. We have chosen the genus Legionella, comprising more than 20 pathogenic species, due to its high relevance for water-based respiratory infections. A new NGS-based approach was designed by sequencing 16S rRNA gene amplicons specific for the genus Legionella using the Illumina MiSeq technology. This approach was validated and applied to a set of representative freshwater samples. Our results revealed that the generated libraries presented a low average raw error rate per base (<0.5%); and substantiated the use of high-fidelity enzymes, such as KAPA HiFi, for increased sequence accuracy and quality. The approach also showed high in situ specificity (>95%) and very good repeatability. Only in samples in which the gammabacterial clade SAR86 was present more than 1% non-Legionella sequences were observed. Next-generation sequencing read counts did not reveal considerable amplification/sequencing biases and showed a sensitive as well as precise quantification of L. pneumophila along a dilution range using a spiked-in, certified genome standard. The genome standard and a mock community consisting of six different Legionella species demonstrated that the developed NGS approach was quantitative and specific at the level of individual species, including L. pneumophila. The sensitivity of our genus-specific approach was at least one order of magnitude higher compared to the universal NGS approach. Comparison of quantification by real-time PCR showed consistency with the NGS data. Overall, our NGS approach can determine the quantitative abundances of Legionella species, i. e. the complete Legionella microbiome, without the need for species-specific primers. The developed NGS approach provides a new molecular surveillance tool to monitor all Legionella species in qualitative and quantitative terms if a spiked-in genome standard is used to calibrate the method. Overall, the genus-specific NGS approach opens up a new avenue to massive parallel diagnostics in a quantitative, specific and sensitive way. The online version of this article (doi:10.1186/s12866-017-0987-5) contains supplementary material, which is available to authorized users.