Serial analysis of ribosomal sequence tags (SARST): a high-throughput method for profiling complex microbial communities
Serial analysis of ribosomal sequence tags (SARST): a high-throughput method for profiling complex microbial communities
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DOI：
10.1046/j.1462-2920.2003.00547.x
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发表时间：
2004-02-01
影响因子：
5.1
通讯作者：
Mohn, WW
中科院分区：
文献类型：
--
作者：
Neufeld, JD;Yu, ZT;Mohn, WW
20年的非培养研究证实，微生物群落代表了地球上最复杂和最集中的系统发育多样性。仍然需要创新的分子工具，可以进一步了解微生物多样性及其功能的影响。我们提出的方法和应用核糖体序列标签（SARST）作为一种新的工具，阐明复杂的微生物群落，如土壤和沉积物中发现的序列分析。核糖体序列标签的系列分析使用一系列酶促反应将核糖体序列标签（RSTs）从细菌小亚基rRNA基因（SSU rDNA）V1-区域扩增并连接到多联体中，所述多联体被克隆和测序。这种方法提供了一个显着增加的吞吐量超过传统的SSU rDNA克隆库，多达20个RST是从每个测序反应。为了测试SARST并测量与这种方法相关的偏差，从纯培养物的定义混合物和重复的北极土壤DNA样品制备了cDNA文库。实际的α分布反映了最初定义的混合物的理论组成。来自重复土壤库的数据（1345和1217个RST，分别有525和505个独特的RST）表明，复制提供了强相关的RSTs分布（r（2）= 0.80）和分区水平分布（r（2）= 0.99）。利用丰富的土壤RSTs的序列数据，我们设计了特异性引物，成功地扩增了更大部分的SSU rDNA进一步的系统发育分析。这些结果表明，SARST是一个强大的方法，可重复的高通量分析的微生物多样性适合医疗，工业或环境微生物学应用。
Two decades of culture-independent studies have confirmed that microbial communities represent the most complex and concentrated pool of phylogenetic diversity on the planet. There remains a need for innovative molecular tools that can further our knowledge of microbial diversity and its functional implications. We present the method and application of serial analysis of ribosomal sequence tags (SARST) as a novel tool for elucidating complex microbial communities, such as those found in soils and sediments. Serial analysis of ribosomal sequence tags uses a series of enzymatic reactions to amplify and ligate ribosomal sequence tags (RSTs) from bacterial small subunit rRNA gene (SSU rDNA) V1-regions into concatemers that are cloned and sequenced. This approach offers a significant increase in throughput over traditional SSU rDNA clone libraries, as up to 20 RSTs are obtained from each sequencing reaction. To test SARST and measure the bias associated with this approach, RST libraries were prepared from a defined mixture of pure cultures and from duplicate arctic soil DNA samples. The actual RST distribution reflected the theoretical composition of the original defined mixture. Data from duplicate soil libraries (1345 and 1217 RSTs, with 525 and 505 unique RSTs, respectively) indicated that replication provides a strongly correlated RST profile (r(2) = 0.80) and division-level distribution of RSTs (r(2) = 0.99). Using sequence data from abundant soil RSTs, we designed specific primers that successfully amplified a larger portion of the SSU rDNA for further phylogenetic analysis. These results suggest that SARST is a powerful approach for reproducible high-throughput profiling of microbial diversity amenable to medical, industrial or environmental microbiology applications.