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首页> 外文期刊>American Journal of Translational Research >The impact of different methods of DNA extraction on microbial community measures of BALF samples based on metagenomic data
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The impact of different methods of DNA extraction on microbial community measures of BALF samples based on metagenomic data

机译：基于宏基因组学数据，不同DNA提取方法对BALF样品微生物群落指标的影响

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Purpose: It is a challenge to find a better microorganisms DNA extraction method for samples taken from the lower airways for metagenomic sequencing, as the concentrations of bacteria in the alveoli and small airways are likely considerably less than that of the mouth or lower digestive tract. Background DNA from the host, and extraction biases can significantly interfere with microbiota assessment and increase the cost of sequencing. This study aimed to develop an optimized DNA extraction method, which would enable a higher concentration of microbial DNA to be extracted from the samples. Methods: We compared the microbiota profiles of the lower airway communities in twelve individuals with IIP. DNA was extracted using three different extraction methods: QIAamp UCP PurePathogen Blood Kit named kit3 in this study, QIAamp UCP Pathogen Mini Kit named kit2, and QIAamp DNA Microbiome Kit named kit1. DNA libraries were constructed according to the manufacturer’s instructions (Illumina). The same workflows from Illumina were used to perform cluster generation, template hybridization, isothermal amplification, linearization, blocking, denaturing, and hybridization of the sequencing primers. Raw data was uploaded to MG-RAST v3 and analyzed. Results: A great number of bacterium inhabits the lower airways of patients with IIP, though there is no airway infection. More bacterium was found in mouth or upper airway. DNA concentrations of DNA samples isolated with kit1 with Benzonase were significantly lower than those isolated with the other two kits for BALF and mouthwash samples. Moreover, the ratio of human genome in clean reads of samples isolated with kit1 with Benzonase was remarkably smaller than those isolated with kit2 and kit3. The relative abundance of total bacteria, the total number of taxa, and the relative abundance of taxa in BALF samples as opposed to mouthwash samples with kit1 were significantly higher than for those extracted the other kits. Conclusion: A microbial DNA extraction method with pretreatment of depletion of host nucleic acid by Benzonase can enable a higher yield of microbial DNA from samples with a higher fraction of host cells to be obtained. The lower airways of patients with IIP without airway infection were inhabited by a great number of bacterium.
机译：目的：对于从下呼吸道采集的样本进行宏基因组测序，找到一种更好的微生物DNA提取方法是一项挑战，因为肺泡和小呼吸道中的细菌浓度可能大大低于口腔或下消化道的细菌浓度。来自宿主的背景DNA和提取偏差会严重干扰微生物群评估，并增加测序成本。这项研究旨在开发一种优化的DNA提取方法，该方法可以从样品中提取更高浓度的微生物DNA。方法：我们比较了十二个患有IIP的个体下呼吸道群落的微生物群分布。使用三种不同的提取方法提取DNA：本研究中名为kit3的QIAamp UCP PurePathogen Blood Kit，名为kit2的QIAamp UCP Pathogen Mini Kit和名为kit1的QIAamp DNA Microbiome Kit。 DNA库是根据制造商的说明（Illumina）构建的。来自Illumina的相同工作流程用于执行测序引物的簇生成，模板杂交，等温扩增，线性化，封闭，变性和杂交。原始数据已上传到MG-RAST v3并进行了分析。结果：尽管没有气道感染，但仍有大量细菌居住在IIP患者的下气道中。在口腔或上呼吸道发现更多细菌。用苯佐那酶kit1分离的DNA样品的DNA浓度明显低于使用其他两种试剂盒分离的BALF和漱口水样品的DNA浓度。而且，用苯佐那酶用kit1分离的样品的干净读数中人基因组的比例明显小于用kit2和kit3分离的样品。与带有kit1的漱口水样品相比，BALF样品中细菌总数，分类单元总数和分类单元的相对丰度显着高于提取其他试剂盒的细菌。结论：采用苯佐那酶预处理微生物核酸的方法对宿主核酸进行了耗尽处理，可以从具有较高宿主细胞比例的样品中获得较高产率的微生物DNA。没有气道感染的IIP患者的下气道被大量细菌占据。

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