Influence of DNA extraction on oral microbial profiles obtained via 16S rRNA gene sequencing.
Influence of DNA extraction on oral microbial profiles obtained via 16S rRNA gene sequencing.
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DOI：
10.3402/jom.v6.23990
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发表时间：
2014
影响因子：
4.5
通讯作者：
Diaz PI
中科院分区：
文献类型：
--
作者：
Abusleme L;Hong BY;Dupuy AK;Strausbaugh LD;Diaz PI
下一代测序的出现极大地促进了口腔微生物组的表征。尽管在简化测序和数据管理过程方面付出了巨大努力，扩增子文库生成所需的上游步骤仍然可能影响基于16S rRNA基因的微生物谱。在上游过程中，DNA提取是一个关键步骤，可能代表一个很大的偏见来源。在比较使用不同方法的研究时，考虑提取程序引入的偏差是很重要的。确定最好地描绘社区的方法也是可取的。因此，本研究的目的是评估不同DNA提取程序对口腔微生物组谱所带来的偏倚。采用4种DNA提取方法对7种具有代表性的口腔细菌模拟群落进行了测试。此外，从7个个体中收集龈上菌斑样本，并将其平均分配以测试两种常用的DNA提取方法。生成16S rRNA基因扩增子文库，并通过454焦磷酸测序进行测序。模拟群落的评估表明，DNA的产量和细菌种类代表随着DNA提取方法的不同而变化。尽管产生的DNA产量最低，但一种包括头部敲击的方法是唯一能够检测模拟群落中所有七个物种的方法。比较两种常用方法（粗解法和化学/酶解+柱基DNA分离法）对菌斑样品的提取效果表明，提取方法对分类群的流行率没有影响，但会影响全球群落结构和单个分类群的相对丰度。在门水平上，后一种方法比粗解法提高了放线菌门、拟杆菌门和螺旋体的回收率。DNA提取会扭曲模拟和临床口腔样本中的微生物图谱，因此需要谨慎选择DNA提取方案，以改善物种恢复并促进口腔微生物学研究中的数据比较。
The advent of next-generation sequencing has significantly facilitated characterization of the oral microbiome. Despite great efforts in streamlining the processes of sequencing and data curation, upstream steps required for amplicon library generation could still influence 16S rRNA gene-based microbial profiles. Among upstream processes, DNA extraction is a critical step that could represent a great source of bias. Accounting for bias introduced by extraction procedures is important when comparing studies that use different methods. Identifying the method that best portrays communities is also desirable. Accordingly, the aim of this study was to evaluate bias introduced by different DNA extraction procedures on oral microbiome profiles. Four DNA extraction methods were tested on mock communities consisting of seven representative oral bacteria. Additionally, supragingival plaque samples were collected from seven individuals and divided equally to test two commonly used DNA extraction procedures. Amplicon libraries of the 16S rRNA gene were generated and sequenced via 454-pyrosequencing. Evaluation of mock communities revealed that DNA yield and bacterial species representation varied with DNA extraction methods. Despite producing the lowest yield of DNA, a method that included bead beating was the only protocol capable of detecting all seven species in the mock community. Comparison of the performance of two commonly used methods (crude lysis and a chemical/enzymatic lysis+column-based DNA isolation) on plaque samples showed no effect of extraction protocols on taxa prevalence but global community structure and relative abundance of individual taxa were affected. At the phylum level, the latter method improved the recovery of Actinobacteria, Bacteroidetes, and Spirochaetes over crude lysis. DNA extraction distorts microbial profiles in simulated and clinical oral samples, reinforcing the importance of careful selection of a DNA extraction protocol to improve species recovery and facilitate data comparison across oral microbiology studies.
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影响因子：
4.2
作者：
Wu GD;Lewis JD;Hoffmann C;Chen YY;Knight R;Bittinger K;Hwang J;Chen J;Berkowsky R;Nessel L;Li H;Bushman FD
通讯作者：
Bushman FD
影响因子：
3.7
作者：
Diaz PI;Dupuy AK;Abusleme L;Reese B;Obergfell C;Choquette L;Dongari-Bagtzoglou A;Peterson DE;Terzi E;Strausbaugh LD
通讯作者：
Strausbaugh LD
影响因子：
3.7
作者：
Willner D;Daly J;Whiley D;Grimwood K;Wainwright CE;Hugenholtz P
通讯作者：
Hugenholtz P
影响因子：
4.4
作者：
Schloss, Patrick D.;Westcott, Sarah L.;Weber, Carolyn F.
通讯作者：
Weber, Carolyn F.
影响因子：
11
作者：
Griffen, Ann L.;Beall, Clifford J.;Leys, Eugene J.
通讯作者：
Leys, Eugene J.