Evaluation of methods to purify virus-like particles for metagenomic sequencing of intestinal viromes.
Evaluation of methods to purify virus-like particles for metagenomic sequencing of intestinal viromes.
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DOI：
10.1186/s12864-014-1207-4
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发表时间：
2015-01-22
期刊：
影响因子：
4.4
通讯作者：
Duerkop BA
中科院分区：
文献类型：
--
作者：
Kleiner M;Hooper LV;Duerkop BA
病毒是哺乳动物肠道微生物群的重要组成部分。近年来，测序技术和数据分析技术的进步使得详细的宏基因组研究能够调查肠道病毒组（噬菌体和真核病毒核酸的集合）及其对微生物群生态的潜在贡献。病毒组研究的一个重要组成部分是从肠道内容物或粪便中分离和纯化病毒样颗粒（VLP）。几种方法已被应用于从肠道样品中分离VLP，但据我们所知，尚未探索方法之间的效率和重现性。对VLP纯化方法的严格评估至关重要，因为许多研究开始从病毒多样性的描述性分析转向努力定量比较许多样本中病毒丰度的研究。因此，需要允许高样品通量的可再现的VLP纯化方法。在这里，我们使用已知细菌和病毒组成的人工肠道微生物群样品比较和评估了四种VLP纯化方法。我们比较了以下四种从粪便样品中纯化VLP的方法：（i）过滤+DNA酶，（ii）二硫苏糖醇处理+过滤+DNA酶，（iii）过滤+DNA酶+ PEG沉淀和（iv）过滤+DNA酶+ CsCl密度梯度离心。四种测试方法中的三种用于VLP纯化效果良好。我们观察到与细菌和宿主DNA的去除效率相关的方法之间的几个差异以及对特定DNA的偏倚。特别是常用于VLP纯化的CsCl密度梯度离心法，在去除宿主来源的DNA方面最有效，但也对特定噬菌体表现出较强的区分力，并且定量结果的重现性较低。根据我们的数据，我们建议在需要对样本间的病毒丰度进行定量比较时，使用方法（i）或（ii）进行大规模研究。CsCl密度梯度离心法虽然非常适合获得高度纯化的样品，但我们认为，在进行定量研究时应谨慎使用。本文的在线版本（doi：10.1186/s12864-014-1207-4）包含补充材料，可供授权用户使用。
Viruses are a significant component of the intestinal microbiota in mammals. In recent years, advances in sequencing technologies and data analysis techniques have enabled detailed metagenomic studies investigating intestinal viromes (collections of bacteriophage and eukaryotic viral nucleic acids) and their potential contributions to the ecology of the microbiota. An important component of virome studies is the isolation and purification of virus-like particles (VLPs) from intestinal contents or feces. Several methods have been applied to isolate VLPs from intestinal samples, yet to our knowledge, the efficiency and reproducibility between methods have not been explored. A rigorous evaluation of methods for VLP purification is critical as many studies begin to move from descriptive analyses of virus diversity to studies striving to quantitatively compare viral abundances across many samples. Therefore, reproducible VLP purification methods which allow for high sample throughput are needed. Here we compared and evaluated four methods for VLP purification using artificial intestinal microbiota samples of known bacterial and viral composition. We compared the following four methods of VLP purification from fecal samples: (i) filtration + DNase, (ii) dithiothreitol treatment + filtration + DNase, (iii) filtration + DNase + PEG precipitation and (iv) filtration + DNase + CsCl density gradient centrifugation. Three of the four tested methods worked well for VLP purification. We observed several differences between methods related to the removal efficiency of bacterial and host DNAs and biases against specific phages. In particular the CsCl density gradient centrifugation method, which is frequently used for VLP purification, was most efficient in removing host derived DNA, but also showed strong discrimination against specific phages and showed a lower reproducibility of quantitative results. Based on our data we recommend the use of methods (i) or (ii) for large scale studies when quantitative comparison of viral abundances across samples is required. The CsCl density gradient centrifugation method, while being excellently suited to achieve highly purified samples, in our opinion, should be used with caution when performing quantitative studies. The online version of this article (doi:10.1186/s12864-014-1207-4) contains supplementary material, which is available to authorized users.
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影响因子：
4.4
作者：
Kim, Min-Soo;Park, Eun-Jin;Bae, Jin-Woo
通讯作者：
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DOI：
10.1073/pnas.1305923110
发表时间：
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影响因子：
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