Efficacy of a 3rd generation high-throughput sequencing platform for analyses of 16S rRNA genes from environmental samples
Efficacy of a 3rd generation high-throughput sequencing platform for analyses of 16S rRNA genes from environmental samples
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DOI：
10.1016/j.mimet.2013.08.009
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发表时间：
2013-11-01
影响因子：
2.2
通讯作者：
Kaplan, Louis A.
中科院分区：
文献类型：
--
作者：
Mosher, Jennifer J.;Bernberg, Erin L.;Kaplan, Louis A.
较长的细菌16 S rRNA基因序列可以提供更大的系统发育和分类分辨率，并在复杂的自然群落中推进种群动态的知识。我们评估了Pacific Biosciences（PacBio）基于DNA聚合的单分子真实的时间（SMRT）测序的准确性，这是一种有前途的第三代高通量技术，并将其与第二代Roche 454焦磷酸测序平台进行了比较。从一个已知的隔离，希瓦氏菌oneidensis MR 1，和环境样品从两个河床栖息地，岩石和沉积物，和河岸带土壤的16 S rRNA基因的扩增子进行了分析。在PacBio上，我们分析了覆盖V1-V3区域的类似500 bp扩增子和V1-V9区域的完整1500 bp扩增子。在Roche 454上，我们分析了类似于500 bp的扩增子。Roche 454方法与分离株相关的错误率最低（2%），PacBio SMRT芯片500 bp扩增子的错误率增加了2倍以上（4-5%），PacBio SMRT芯片全基因的错误率增加了8倍以上（17与18%相似）。PacBio SMRT芯片的较高错误率人为地夸大了对丰富度的估计，并降低了对环境样本覆盖率的估计。我们评估的第三代测序技术没有为微生物生态学研究提供更高的系统发育和分类分辨率。(C)2013爱思唯尔有限公司版权所有。
Longer sequences of the bacterial 16S rRNA gene could provide greater phylogenetic and taxonomic resolutions and advance knowledge of population dynamics within complex natural communities. We assessed the accuracy of a Pacific Biosciences (PacBio) single molecule, real time (SMRT) sequencing based on DNA polymerization, a promising 3rd generation high-throughput technique, and compared this to the 2nd generation Roche 454 pyrosequencing platform. Amplicons of the 16S rRNA gene from a known isolate, Shewanella oneidensis MR1, and environmental samples from two streambed habitats, rocks and sediments, and a riparian zone soil, were analyzed. On the PacBio we analyzed similar to 500 bp amplicons that covered the V1-V3 regions and the full 1500 bp amplicons of the V1-V9 regions. On the Roche 454 we analyzed the similar to 500 bp amplicons. Error rates associated with the isolate were lowest with the Roche 454 method (2%), increased by more than 2-fold for the 500 bp amplicons with the PacBio SMRT chip (4-5%), and by more than 8-fold for the full gene with the PacBio SMRT chip (17 similar to 18%). Higher error rates with the PacBio SMRT chip artificially inflated estimates of richness and lowered estimates of coverage for environmental samples. The 3rd generation sequencing technology we evaluated does not provide greater phylogenetic and taxonomic resolutions for studies of microbial ecology. (C) 2013 Elsevier B.V. All rights reserved.