Exploring microbial diversity and taxonomy using SSU rRNA hypervariable tag sequencing.
Exploring microbial diversity and taxonomy using SSU rRNA hypervariable tag sequencing.
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DOI：
10.1371/journal.pgen.1000255
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发表时间：
2008-11
期刊：
影响因子：
4.5
通讯作者：
Sogin ML
中科院分区：
文献类型：
--
作者：
Huse SM;Dethlefsen L;Huber JA;Mark Welch D;Relman DA;Sogin ML
小亚基核糖体RNA（SSU rRNA）基因的高变区的大规模平行焦磷酸测序可以比全长SSU rRNA的毛细管测序每美元和每小时更深地采样微生物群落两个或三个数量级。与全长rRNA调查一样，每个序列读数都是单个微生物的标签替代物。然而，而不是通过从头创建包括所有实验序列和某些参考分类群的基因树来分配分类法，我们将高变区标签与广泛的rRNA序列数据库进行比较，并基于全局序列比对分类法（GAST）过程中的最佳匹配来分配分类法。由此产生的分类普查提供了微生物群落的组成和多样性的信息。为了确定仅使用高变区标签评估微生物群落成员的有效性，我们将分配给V3和V6高变区的分类与分配给从人类肠道和深海热液喷口分离的全长SSU rRNA序列的分类进行了比较。高变区标签和全长rRNA序列提供了等效的微生物群落的相对丰度的分类和措施，即使标签高达15%的分歧，从他们最近的参考匹配。大规模并行焦磷酸测序提供的每美元更大的采样深度比全长基因的毛细管测序揭示了更多的“稀有生物圈”成员。此外，标签测序消除了克隆偏倚，并且序列足够短，可以在单次读取中完全测序，从而最大限度地增加运行中采样的生物体数量，同时最大限度地减少嵌合体形成。该技术允许以具有成本效益的方式探索微生物群落结构的变化，包括罕见的生物圈，随着空间和时间的推移，并可立即应用于人类微生物组项目等举措。微生物在人类和环境健康中起着至关重要的作用。我们越是探索微生物种群，我们发现的复杂性和多样性就越多。基于16 S核糖体RNA基因的系统发育树已被成功地用于仅从DNA鉴定微生物分类。新的DNA测序技术，如大规模并行焦磷酸测序，可以提供比以往更多数量级的DNA序列，然而，序列要短得多，因此需要新的方法来从短DNA标签中识别微生物。我们证明了识别微生物类群的有效性，通过比较短的标签从16 S高变区对一个大的数据库中已知的16 S基因。使用这种技术，高变区标签提供了与全长rRNA序列相当的微生物群落分类和相对丰度。标签焦磷酸测序提供的更大的采样深度不仅揭示了占主导地位的微生物物种，而且比全长基因的毛细管测序更多的“稀有生物圈”成员。标签焦磷酸测序极大地增强了探索微生物种群组成、多样性和分布的项目，如人类微生物组计划。《公共科学图书馆生物学》的一篇配套论文（见Dethlefsen et al.，DOI：10.1371/journal.pbio.0060280）成功地使用该技术来表征抗生素对人肠道微生物群的作用。
Massively parallel pyrosequencing of hypervariable regions from small subunit ribosomal RNA (SSU rRNA) genes can sample a microbial community two or three orders of magnitude more deeply per dollar and per hour than capillary sequencing of full-length SSU rRNA. As with full-length rRNA surveys, each sequence read is a tag surrogate for a single microbe. However, rather than assigning taxonomy by creating gene trees de novo that include all experimental sequences and certain reference taxa, we compare the hypervariable region tags to an extensive database of rRNA sequences and assign taxonomy based on the best match in a Global Alignment for Sequence Taxonomy (GAST) process. The resulting taxonomic census provides information on both composition and diversity of the microbial community. To determine the effectiveness of using only hypervariable region tags for assessing microbial community membership, we compared the taxonomy assigned to the V3 and V6 hypervariable regions with the taxonomy assigned to full-length SSU rRNA sequences isolated from both the human gut and a deep-sea hydrothermal vent. The hypervariable region tags and full-length rRNA sequences provided equivalent taxonomy and measures of relative abundance of microbial communities, even for tags up to 15% divergent from their nearest reference match. The greater sampling depth per dollar afforded by massively parallel pyrosequencing reveals many more members of the “rare biosphere” than does capillary sequencing of the full-length gene. In addition, tag sequencing eliminates cloning bias and the sequences are short enough to be completely sequenced in a single read, maximizing the number of organisms sampled in a run while minimizing chimera formation. This technique allows the cost-effective exploration of changes in microbial community structure, including the rare biosphere, over space and time and can be applied immediately to initiatives, such as the Human Microbiome Project. Microbes play a critical role in both human and environmental health. The more we explore microbial populations, the more complexity and diversity we find. Phylogenetic trees based on 16S ribosomal RNA genes have been used with great success to identify microbial taxonomy from DNA alone. New DNA sequencing technologies, such as massively parallel pyrosequencing, can provide orders of magnitude more DNA sequences than ever before, however, the sequences are much shorter, so new methods are necessary to identify the microbes from short DNA tags. We demonstrate the effectiveness of identifying microbial taxa by comparing short tags from 16S hypervariable regions against a large database of known 16S genes. Using this technique, hypervariable region tags provide equivalent taxonomy and relative abundances of microbial communities as full-length rRNA sequences. The greater sampling depth afforded by tag pyrosequencing uncovers not only the dominant microbial species, but many more members of the “rare biosphere” than does capillary sequencing of the full-length gene. Tag pyrosequencing greatly enhances projects exploring composition, diversity, and distribution of microbial populations, such as the Human Microbiome Initiative. A companion paper in PLoS Biology (see Dethlefsen et al., doi:10.1371/journal.pbio.0060280) successfully uses this technique to characterize the effects of antibiotics on the human gut microbiota.
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影响因子：
14.9
作者：
Ludwig, W;Strunk, O;Schleifer, KH
通讯作者：
Schleifer, KH
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：
10.1073/pnas.0608255104
发表时间：
2007-01-02
影响因子：
11.1
作者：
Brodie, Eoin L.;DeSantis, Todd Z.;Andersen, Gary L.
通讯作者：
Andersen, Gary L.
影响因子：
14.9
作者：
Liu Z;Lozupone C;Hamady M;Bushman FD;Knight R
通讯作者：
Knight R
影响因子：
56.9
作者：
Huber, Julie A.;Mark Welch, David;Sogin, Mitchell L.
通讯作者：
Sogin, Mitchell L.