Reproducibility and quantitation of amplicon sequencing-based detection

被引：341

作者：

Zhou, Jizhong ^{[1
,2
,3
]}

Wu, Liyou ^{[1
]}

Deng, Ye ^{[1
]}

Zhi, Xiaoyang ^{[1
]}

Jiang, Yi-Huei ^{[1
]}

Tu, Qichao ^{[1
]}

Xie, Jianping ^{[1
]}

Van Nostrand, Joy D. ^{[1
]}

He, Zhili ^{[1
]}

Yang, Yunfeng ^{[2
]}

机构：

[1] Univ Oklahoma, Dept Bot & Microbiol, Inst Environm Genom, Norman, OK 73019 USA

[2] Tsinghua Univ, Dept Environm Sci & Engn, Beijing 100084, Peoples R China

[3] Univ Calif Berkeley, Lawrence Berkeley Lab, Div Earth Sci, Berkeley, CA 94720 USA

来源：

ISME JOURNAL | 2011年 / 5卷 / 08期

基金：

美国农业部;

关键词：

MICROBIAL COMMUNITY COMPOSITION; BACTERIAL DIVERSITY; SPECIES RICHNESS; RARE BIOSPHERE; SOIL; REVEALS; GENES; AMPLIFICATION; METAGENOMICS; RHIZOSPHERE;

D O I：

10.1038/ismej.2011.11

中图分类号：

Q14 [生态学（生物生态学）];

学科分类号：

071012 ; 0713 ;

摘要：

To determine the reproducibility and quantitation of the amplicon sequencing-based detection approach for analyzing microbial community structure, a total of 24 microbial communities from a long-term global change experimental site were examined. Genomic DNA obtained from each community was used to amplify 16S rRNA genes with two or three barcode tags as technical replicates in the presence of a small quantity (0.1% wt/wt) of genomic DNA from Shewanella oneidensis MR-1 as the control. The technical reproducibility of the amplicon sequencing-based detection approach is quite low, with an average operational taxonomic unit (OTU) overlap of 17.2%+/-2.3% between two technical replicates, and 8.2%+/-2.3% among three technical replicates, which is most likely due to problems associated with random sampling processes. Such variations in technical replicates could have substantial effects on estimating beta-diversity but less on alpha-diversity. A high variation was also observed in the control across different samples (for example, 66.7-fold for the forward primer), suggesting that the amplicon sequencing-based detection approach could not be quantitative. In addition, various strategies were examined to improve the comparability of amplicon sequencing data, such as increasing biological replicates, and removing singleton sequences and less-representative OTUs across biological replicates. Finally, as expected, various statistical analyses with preprocessed experimental data revealed clear differences in the composition and structure of microbial communities between warming and non-warming, or between clipping and non-clipping. Taken together, these results suggest that amplicon sequencing-based detection is useful in analyzing microbial community structure even though it is not reproducible and quantitative. However, great caution should be taken in experimental design and data interpretation when the amplicon sequencing-based detection approach is used for quantitative analysis of the beta-diversity of microbial communities. The ISME Journal (2011) 5, 1303-1313; doi:10.1038/ismej.2011.11; published online 24 February 2011

引用

页码：1303 / 1313

页数：11

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