Optimizing protocols for extraction of bacteriophages prior to metagenomic analyses of phage communities in the human gut. Extraction of bacteriophages from fecal samples

Background: The human gut is densely populated with archaea, eukaryotes, bacteria and their viruses, such as bacteriophages, that play also a prominent role. Recent 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 communities prior to HTS and their impact on the metagenomic dataset. Results: 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 (LIT). To quantify phage recovery, samples were spiked with c2, ϕ29 and T4 phages (representatives of the Siphoviridae, Podoviridae and Myoviridae families, respectively) and their concentration (determined as plaque forming units, PFU) followed at every step during the extraction procedure. Compared with LIT, TFF and PEG had higher recovery rates of all spiked phages, yielding up to 16 times more phage particles (PPs) and up to 68 times more phage DNA per volume. By improving phage extraction, the metagenomic dataset showed a 10% increase in relative distribution of Caudovirales and unclassified phages infecting gut-associated bacteria (16%) as compared to the reference protocol (27%). This latter decline, however, was not considered as Myophages loss, but rather an increase in the abundance of Siphophages (TFF and PEG: <36.8%, LIT: 28.4%), whose greater extraction, was achieved through to the enhanced filtration conditions of our procedures (e.g. reduction of filter clogging). Conclusion: Two procedures for extraction of bacteriophages from fecal samples, relaying on two well known laboratory techniques such as TFF and PEG, were optimized and standardized using a set of spiked bacteriophages as process control. These optimized procedures proved to be highly efficient and robust tools for extraction and purification of PPs that are suitable for quantification, high-throughput sequencing and further phageome studies.

背景：人体肠道内定植有古菌、真核生物、细菌及其病毒（如噬菌体），其中噬菌体亦发挥着重要作用。近年来，高通量测序（high-throughput sequencing, HTS）与生物信息学（bioinformatics）技术的发展为解析肠道病毒群落提供了新的契机。然而在高通量测序技术应用之前，学界对这类肠道病毒群落及其对宏基因组（metagenomic）数据集的影响关注度有限。

结果：本研究基于切向流过滤（tangential-flow filtration, TFF）与聚乙二醇沉淀（polyethylene glycol precipitation, PEG）技术，开发了两种粪便样本噬菌体提取优化方法，并以已发表方案的改编版本作为对照（LIT）。为定量噬菌体的提取回收率，我们向样本中加入了分别代表长尾噬菌体科（Siphoviridae）、短尾噬菌体科（Podoviridae）与肌尾噬菌体科（Myoviridae）的c2、ϕ29及T4噬菌体，并在提取流程的每一步以噬菌斑形成单位（plaque forming units, PFU）为指标检测其浓度。与对照LIT相比，TFF与PEG法对所有加标噬菌体的回收率更高，每单位体积可获得的噬菌体颗粒（phage particles, PPs）数量最多提升16倍，噬菌体DNA总量最多提升68倍。通过优化噬菌体提取流程，宏基因组数据集中感染肠道相关细菌的有尾噬菌体目（Caudovirales）与未分类噬菌体的相对占比较参照方案提升10%，优化组占比为16%，参照方案组为27%。不过这一相对占比的差异并非源于肌尾噬菌体（Myophages）的流失，而是由于长尾噬菌体（Siphophages）丰度提升：TFF与PEG法中长尾噬菌体占比低于36.8%，而LIT法中该占比为28.4%——本研究通过优化过滤条件（例如减少过滤器堵塞）实现了更高的噬菌体提取效率。

结论：本研究以一系列加标噬菌体作为流程对照，对两种基于经典实验室技术（TFF与PEG）的粪便样本噬菌体提取方法进行了优化与标准化。经优化的两种方法可高效、稳定地提取与纯化噬菌体颗粒（PPs），适用于噬菌体定量、高通量测序及后续噬菌体组（phageome）研究。