ena-STUDY-UNIVERSITY OF COPENHAGEN-02-02-2015-11:40:08:462-77
Optimizing protocols for extraction of bacteriophages prior to metagenomic analyses of phage communities in the human gut
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, ?29 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  (<92% for TFF and PEG; while 82.4% for LIT). TFF and PEG methods obtained a lower relative abundance of the Myoviridae family (>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.
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, ?29 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  (<92% for TFF and PEG; while 82.4% for LIT). TFF and PEG methods obtained a lower relative abundance of the Myoviridae family (>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.