Bacterial flora-typing with targeted, chip-based Pyrosequencing
Bacterial flora-typing with targeted, chip-based Pyrosequencing
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DOI：
10.1186/1471-2180-7-108
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发表时间：
2007-11-30
期刊：
影响因子：
4.2
通讯作者：
Ronaghi, Mostafa
中科院分区：
文献类型：
--
作者：
Sundquist, Andreas;Bigdeli, Saharnaz;Ronaghi, Mostafa
背景：微生物群落的宏基因组分析有可能提高我们对微生物在临床条件中的作用的理解。最近，DNA测序通量和成本的显著提高将使对个体的分析成为可能。然而，这种吞吐量的提高通常是以更短的读取长度为代价的，这限制了每次读取的区分能力。特别是，通过< 1,600 bp 16S rRNA基因测序对样品微生物含量进行分类将受到这种限制的影响。结果：我们描述了一种利用针对16S rRNA基因的高通量焦磷酸测序来鉴定细菌样本系统发育内容的方法。我们的分析适用于这种技术的较短的读取长度，并使用16S rDNA数据库来确定读取的最具体的系统发育分类，从而得出一个加权的系统发育树来表征样品的内容。我们目前的结果，从人类阴道在怀孕期间获得的六个样本，证实了以前的研究使用传统技术。接下来，我们通过模拟实验分析了我们的方法在系统发育的每个层次上对reads进行分类的能力。我们评估了读取长度和数据库完整性对我们方法的影响，并预测随着技术的进步和更多细菌的测序，我们将如何做。最后，我们研究了靶向特定16S可变区域的效用，并表明这种方法大大提高了某些类型微生物样品的结果。通过仿真，我们的方法可以用来确定信息量最大的变量区域。结论：本研究为利用短读测序技术靶向16S宏基因组的有效性提供了积极的验证。我们的方法使我们能够推断出系统发育中最具体的序列分配，并确定最具歧视性的变量区域。对人类菌群样本进行高通量焦磷酸测序分析，将加速研究微生物世界与人类之间的关系。
Background: The metagenomic analysis of microbial communities holds the potential to improve our understanding of the role of microbes in clinical conditions. Recent, dramatic improvements in DNA sequencing throughput and cost will enable such analyses on individuals. However, such advances in throughput generally come at the cost of shorter read-lengths, limiting the discriminatory power of each read. In particular, classifying the microbial content of samples by sequencing the < 1,600 bp 16S rRNA gene will be affected by such limitations.Results: We describe a method for identifying the phylogenetic content of bacterial samples using high-throughput Pyrosequencing targeted at the 16S rRNA gene. Our analysis is adapted to the shorter read-lengths of such technology and uses a database of 16S rDNA to determine the most specific phylogenetic classification for reads, resulting in a weighted phylogenetic tree characterizing the content of the sample. We present results for six samples obtained from the human vagina during pregnancy that corroborates previous studies using conventional techniques.Next, we analyze the power of our method to classify reads at each level of the phylogeny using simulation experiments. We assess the impacts of read-length and database completeness on our method, and predict how we do as technology improves and more bacteria are sequenced. Finally, we study the utility of targeting specific 16S variable regions and show that such an approach considerably improves results for certain types of microbial samples. Using simulation, our method can be used to determine the most informative variable region.Conclusion: This study provides positive validation of the effectiveness of targeting 16S metagenomes using short-read sequencing technology. Our methodology allows us to infer the most specific assignment of the sequence reads within the phylogeny, and to identify the most discriminative variable region to target. The analysis of high-throughput Pyrosequencing on human flora samples will accelerate the study of the relationship between the microbial world and ourselves.