Comparison of DNA extraction methods for microbial community profiling with an application to pediatric bronchoalveolar lavage samples.
Comparison of DNA extraction methods for microbial community profiling with an application to pediatric bronchoalveolar lavage samples.
复制标题
DOI：
10.1371/journal.pone.0034605
复制
发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Hugenholtz P
中科院分区：
文献类型：
--
作者：
Willner D;Daly J;Whiley D;Grimwood K;Wainwright CE;Hugenholtz P
条形码扩增子测序正迅速成为分析微生物群落（包括人类呼吸道微生物组）的标准方法。虽然这种方法比标准培养具有更小的偏差，但几个步骤可以引入变化，包括所使用的DNA提取方法的类型。在这里，我们评估了五种不同的提取方法对小儿支气管肺泡灌洗（BAL）样本和模拟社区组成的9个细菌属，以确定这些典型的低复杂性社区的方法重现性和检测限。此外，使用模拟社区，我们能够评估污染，并选择一个相对丰度截止阈值的基础上的几何分布，优化之间的权衡检测真正的操作分类单位和过滤出虚假的。使用该阈值，通过所有提取方法可预测地检测到模拟群落中的大多数属，包括难以裂解的革兰氏阳性属葡萄球菌。对于模拟群落和BAL样本，提取方法之间的差异显着大于技术重复之间的差异，这强调了使用标准化方法进行微生物组研究的重要性。然而，无论使用何种方法，个体患者都保留了独特的诊断特征。此外，尽管作为原始冷冻样品储存超过5年，但BAL样品的群落特征与历史培养结果一致。这些样本的非培养分析还确定了许多厌氧属，这些属被认为是呼吸道微生物组的一部分。这项研究应该有助于指导研究人员制定呼吸道和其他人体微生物组样本的采样，提取和分析策略。
Barcoded amplicon sequencing is rapidly becoming a standard method for profiling microbial communities, including the human respiratory microbiome. While this approach has less bias than standard cultivation, several steps can introduce variation including the type of DNA extraction method used. Here we assessed five different extraction methods on pediatric bronchoalveolar lavage (BAL) samples and a mock community comprised of nine bacterial genera to determine method reproducibility and detection limits for these typically low complexity communities. Additionally, using the mock community, we were able to evaluate contamination and select a relative abundance cut-off threshold based on the geometric distribution that optimizes the trade off between detecting bona fide operational taxonomic units and filtering out spurious ones. Using this threshold, the majority of genera in the mock community were predictably detected by all extraction methods including the hard-to-lyse Gram-positive genus Staphylococcus. Differences between extraction methods were significantly greater than between technical replicates for both the mock community and BAL samples emphasizing the importance of using a standardized methodology for microbiome studies. However, regardless of method used, individual patients retained unique diagnostic profiles. Furthermore, despite being stored as raw frozen samples for over five years, community profiles from BAL samples were consistent with historical culturing results. The culture-independent profiling of these samples also identified a number of anaerobic genera that are gaining acceptance as being part of the respiratory microbiome. This study should help guide researchers to formulate sampling, extraction and analysis strategies for respiratory and other human microbiome samples.
登录
查看更多内容
DOI：
10.1093/bioinformatics/btq365
发表时间：
2010-09-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Balzer S;Malde K;Lanzén A;Sharma A;Jonassen I
通讯作者：
Jonassen I
影响因子：
3.7
作者：
Erb-Downward JR;Thompson DL;Han MK;Freeman CM;McCloskey L;Schmidt LA;Young VB;Toews GB;Curtis JL;Sundaram B;Martinez FJ;Huffnagle GB
通讯作者：
Huffnagle GB
影响因子：
2.1
作者：
Grahn, N;Olofsson, M;Jonasson, J
通讯作者：
Jonasson, J
影响因子：
11
作者：
Guss, Adam M.;Roeselers, Guus;Cavanaugh, Colleen M.
通讯作者：
Cavanaugh, Colleen M.
DOI：
10.1073/pnas.1002601107
发表时间：
2010-06-29
影响因子：
11.1
作者：
Dominguez-Bello, Maria G.;Costello, Elizabeth K.;Knight, Rob
通讯作者：
Knight, Rob