Optimizing protocols for extraction of bacteriophages prior to metagenomic analyses of phage communities in the human gut.
Optimizing protocols for extraction of bacteriophages prior to metagenomic analyses of phage communities in the human gut.
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DOI：
10.1186/s40168-015-0131-4
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发表时间：
2015-11-17
期刊：
影响因子：
15.5
通讯作者：
Nielsen DS
中科院分区：
文献类型：
--
作者：
Castro-Mejía JL;Muhammed MK;Kot W;Neve H;Franz CM;Hansen LH;Vogensen FK;Nielsen DS
人类肠道中密集分布着古细菌、真核生物、细菌及其病毒，如噬菌体。高通量测序（HTS）和生物信息学的进步为表征肠道中的病毒群落提供了新的机会。然而，有限的注意力已经给了处理从粪便群落提取的粪便中的HTS之前，其对宏基因组数据集的影响的协议的效率。我们描述了两种优化的方法，用于从粪便样品中提取粪便，基于切向流过滤（TFF）和聚乙二醇沉淀（PEG）方法，使用已发表的方案作为对照（文献改编方案（LIT））的改编方法。为了定量噬菌体回收率，在提取过程的每个步骤中，向样品中掺入少量的c2、c29和T4 α（分别代表Siphoviridae、Podoviridae和Myoviridae家族）及其浓度（噬斑形成单位）。与LIT相比，TFF和PEG具有更高的回收率，每体积产生高达16倍的噬菌体颗粒（PP）和高达68倍的噬菌体DNA，从而增加了提取低丰度噬菌体的机会。TFF和PEG衍生的偏病毒组显示尾病毒目和未分类的感染肠道相关细菌的细菌的相对丰度增加10%（TFF和PEG> 92%，LIT> 82.4%）。与参考方案（22%）相比，我们的方法获得了较低的肌尾病毒科（Myoviridae）家族的相对丰度（<16%）。然而，这种下降不被认为是肌尾病毒科的真正损失，而是更高水平的虹吸病毒科的提取（TFF和PEG > 32.5%，LIT 22.6%），这是在我们的方法的增强条件下实现的（例如，减少过滤器堵塞）。通过透射电子显微镜记录了使用TFF和PEG提取的样品中的高度噬菌体多样性。使用一组加标噬菌体作为过程控制，优化了从粪便样品中提取噬菌体的两种方法（TFF和PEG）。这些方案是用于在噬菌体-元病毒组研究中HTS之前提取和纯化PP的高效工具。我们的方法可以很容易地修改，因此原则上适用于任何溶解的固体环境材料。本文的在线版本（doi：10.1186/s40168-015-0131-4）包含补充材料，可供授权用户使用。
The human gut is densely populated with archaea, eukaryotes, bacteria, and their viruses, such as bacteriophages. Advances in high-throughput sequencing (HTS) as well as bioinformatics have opened new opportunities for characterizing the viral communities harbored in our gut. However, limited attention has been given to the efficiency of protocols dealing with extraction of phages from fecal communities prior to HTS and their impact on the metagenomic dataset. We describe two optimized methods for extraction of phages from fecal samples based on tangential-flow filtration (TFF) and polyethylene glycol precipitation (PEG) approaches using an adapted method from a published protocol as control (literature-adapted protocol (LIT)). To quantify phage recovery, samples were spiked with low numbers of c2, ϕ29, and T4 phages (representatives of the Siphoviridae, Podoviridae, and Myoviridae families, respectively) and their concentration (plaque-forming units) followed at every step during the extraction procedure. Compared with LIT, TFF and PEG had higher recovery of all spiked phages, yielding up to 16 times more phage particles (PPs) and up to 68 times more phage DNA per volume, increasing thus the chances of extracting low abundant phages. TFF- and PEG-derived metaviromes showed 10 % increase in relative abundance of Caudovirales and unclassified phages infecting gut-associated bacteria (>92 % for TFF and PEG, 82.4 % for LIT). Our methods obtained lower relative abundance of the Myoviridae family (<16 %) as compared to the reference protocol (22 %). This decline, however, was not considered a true loss of Myoviridae phages but rather a greater level of extraction of Siphoviridae phages (TFF and PEG >32.5 %, LIT 22.6 %), which was achieved with the enhanced conditions of our procedures (e.g., reduced filter clogging). A high degree of phage diversity in samples extracted using TFF and PEG was documented by transmission electron microscopy. Two procedures (TFF and PEG) for extraction of bacteriophages from fecal samples were optimized using a set of spiked bacteriophages as process control. These protocols are highly efficient tools for extraction and purification of PPs prior to HTS in phage-metavirome studies. Our methods can be easily modified, being thus applicable and adjustable for in principle any solid environmental material in dissolution. The online version of this article (doi:10.1186/s40168-015-0131-4) contains supplementary material, which is available to authorized users.