Department of Agroecology, Faculty of Science and Technology, Aarhus University, Forsøgsvej 1, 4200 Slagelse, Denmark.
Can J Microbiol. 2011 Dec;57(12):1062-6. doi: 10.1139/w11-100. Epub 2011 Dec 1.
Most studies involving next-generation amplicon sequencing of microbial communities from environmental studies lack replicates. DNA extraction and PCR effects on the variation of read abundances of operational taxonomic units generated from deep amplicon 454 pyrosequencing was investigated using soil samples from an agricultural field with diseased pea. One sample was extracted four times, and one of these samples was PCR amplified four times to obtain eight replicates in total. Results showed that species richness was consistent among replicates. Variation among dominant taxa was low across replicates, whereas rare operational taxonomic units showed higher variation among replicates. The results indicate that pooling of several extractions and PCR amplicons will decrease variation among samples.
大多数涉及环境研究中微生物群落的下一代扩增子测序的研究都缺乏重复。本研究使用来自豌豆病害农田的土壤样本，调查了 DNA 提取和 PCR 对 454 焦磷酸测序深度扩增子产生的操作分类单位的读长丰度的变化。一个样本被提取了四次，其中一个样本被 PCR 扩增了四次，总共得到了 8 个重复。结果表明，重复样本中的物种丰富度是一致的。在重复样本中，优势分类群之间的变异很小，而稀有操作分类单元的变异较大。结果表明，汇集多个提取和 PCR 扩增子将降低样本之间的变异。
