Bias in bacterial diversity as a result of Nycodenz extraction from bulk soil
Bias in bacterial diversity as a result of Nycodenz extraction from bulk soil
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DOI：
10.1016/j.soilbio.2011.06.019
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发表时间：
2011-10-01
影响因子：
9.7
通讯作者：
Sorensen, Soren J.
中科院分区：
文献类型：
--
作者：
Holmsgaard, Peter N.;Norman, Anders;Sorensen, Soren J.
Nycodenz密度离心法(NDC)是一种分离方法，可以从土壤中提取可培养和不可培养的细菌细胞，用于进一步的下游分析；然而，到目前为止，关于这种方法的效率缺乏信息。因此，这项研究的目的是调查从土壤中提取NDC的总体效率，并确定采样偏差(如果有的话)。使用已经建立的NDC方案从丹麦关键田间试验的三个土块中提取细菌细胞。为了评估NDC程序的各个方面，直接从土壤中提取DNA，从NDC提取的细胞中提取DNA，以及从NDC后留下的土壤颗粒中提取DNA。从提取的DNA中扩增16S rRNA的V4-V6区，然后使用454 Genome Sequencer FLX系统进行样品标记扩增-焦磷酸测序，以评估细菌多样性。使用核糖体数据库项目(RDP)的焦糖测序管道工具对序列进行处理和分析。在这项研究中，我们证明了使用NDC从土壤中提取细菌会导致显著的偏差，表现为土壤中常见的特定细菌门的过度表达或表达不足。稀疏性分析、相似性分析、多维标度图和方差分析表明，NDC提取的样品与原始土壤样品和剩余的NDC颗粒样品相比，多样性都显著降低。为了进一步研究土壤多样性，一个数学模型被用来估计需要多少序列才能找到土壤中95%的可操作分类单元(OTU)。该模型估计土壤中含有大约29,400个OTU，只需要351,500个序列就可以覆盖95%的细菌生物多样性，相当于一次完整的标准GS FLX运行。(C)2011爱思唯尔有限公司。保留所有权利。
Nycodenz density centrifugation (NDC) is an isolation method that allows extraction of both culturable and unculturable bacterial cells from soil, to be used in further downstream analysis; however, to date there has been a lack of information concerning the efficiency of this method. The aim of this study was therefore to investigate the overall efficiency of NDC extractions from soil and to identify sampling bias, if any.Bacterial cells were extracted from three soil plots from the Danish CRUCIAL field trial using an already established NDC protocol. To evaluate all aspects of the NDC procedure, DNA was extracted directly from soil, from NDC-extracted cells, and from the soil pellets left after NDC. Bacterial diversity was assessed by PCR amplification of the V4-V6 regions of the 16S rRNA from the extracted DNA followed by sample-tagged amplicon-pyrosequencing using the 454 Genome Sequencer FLX system. Sequences were processed and analyzed using the Ribosomal Database Project's (RDP) Pyrosequencing Pipeline tools.In this study, we show that extraction of bacteria from soil using NDC can result in significant biases in the form of either over- or underrepresentation of specific bacterial phyla commonly found in soil. Furthermore, rarefaction analysis, analysis of similarity, multidimensional scaling plots and analysis of variance showed that the diversity in the NDC-extracted sample was reduced significantly compared to both the original soil sample and the remaining NDC-pellet. To further study the soil diversity a mathematical model was employed to estimate how many sequences would be required in order to find 95% of all operational taxonomic units (OTUs) in the soil. The model estimated that the soil contains approximately 29,400 OTUs and that just 351,500 sequences are needed to cover 95% of the bacterial biodiversity, the equivalent of one full standard GS FLX run. (C) 2011 Elsevier Ltd. All rights reserved.