Effect of PCR amplicon size on assessments of clone library microbial diversity and community structure

被引:109
作者
Huber, Julie A. [1 ]
Morrison, Hilary G. [1 ]
Huse, Susan M. [1 ]
Neal, Phillip R. [1 ]
Sogin, Mitchell L. [1 ]
Welch, David B. Mark [1 ]
机构
[1] Marine Biol Lab, Josephine Bay Paul Ctr, Woods Hole, MA 02543 USA
基金
美国国家科学基金会; 美国国家卫生研究院;
关键词
16S RIBOSOMAL-RNA; POLYMERASE-CHAIN-REACTION; ANNEALING TEMPERATURE; MULTITEMPLATE PCR; PRIMER MISMATCH; GENES; BIAS; AMPLIFICATION; HETERODUPLEXES; ENVIRONMENT;
D O I
10.1111/j.1462-2920.2008.01857.x
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
PCR-based surveys of microbial communities commonly use regions of the small-subunit ribosomal RNA (SSU rRNA) gene to determine taxonomic membership and estimate total diversity. Here we show that the length of the target amplicon has a significant effect on assessments of microbial richness and community membership. Using operational taxonomic unit (OTU)- and taxonomy-based tools, we compared the V6 hypervariable region of the bacterial SSU rRNA gene of three amplicon libraries of c. 100, 400 and 1000 base pairs (bp) from each of two hydrothermal vent fluid samples. We found that the smallest amplicon libraries contained more unique sequences, higher diversity estimates and a different community structure than the other two libraries from each sample. We hypothesize that a combination of polymerase dissociation, cloning bias and mispriming due to secondary structure accounts for the differences. While this relationship is not linear, it is clear that the smallest amplicon libraries contained more different types of sequences, and accordingly, more diverse members of the community. Because divergent and lower abundant taxa can be more readily detected with smaller amplicons, they may provide better assessments of total community diversity and taxonomic membership than longer amplicons in molecular studies of microbial communities.
引用
收藏
页码:1292 / 1302
页数:11
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